Nogo, caspr, f3 nb-3 useful in the treatment of injury and disease to the central nervous system

ABSTRACT

The application provides materials and methods for promoting myelination of neuronal axons in the CNS. These derive from the findings firstly that the molecules Nogo and Caspr interact with one another during establishment and maintenance of the axoglial junction, and secondly that the molecules F3 and NB-3 are capable of promoting oligodendrocyte maturation via interaction with Notch. The materials and methods provided may be used in the treatment of CNS damage, in particular the treatment of spinal cord injury, multiple sclerosis, epilepsy and stroke.

FIELD OF THE INVENTION

The present invention relates to novel methods and materials fortreating injury and disease of the central nervous system (CNS).Particularly, but not exclusively, the invention provides methods andmaterials for spinal cord regeneration and remyelination followingspinal cord injury (SCI), stroke, or disease such as multiple sclerosis(MS) and epilepsy.

BACKGROUND TO THE INVENTION

Over 250,000 people in the United States, and several million wordwide,are permanently disabled due to a past spinal cord injury (chronic SCI),and about 12,000 people are newly injured (acute SCI) in the UnitedStates each year. Additionally, paralysis due to SCI is predominantly acondition of the young: 60% of spinal cord injuries occur before age 30,and the most frequent incidence is at age 19. Most injuries are causedby motor vehicle, sports or work-related accidents, or by violence.Estimated costs of care for SCI patients in the United States aloneexceed $9 billion per year and over $1.5 million per patient lifetime.

Following trauma to the adult central nervous system (CNS) of mammals,injured neurons do not regenerate their transected axons. Currently, themainstay of treatment in spinal cord injury is still rehabilitation.There is little one can do to address the primary injury. After spinalinjury, three major classes of damage have been identified:

-   1. Neuronal cell death. The death of nerve cells due to injury    presents a difficult problem because nerve cells lose the ability to    undergo cell division as they mature into the highly specialized    cells that make up our nervous systems. Some cells die during the    traumatic insult, others die in the hours, days or even weeks    following injury. Regardless of when the cell death occurs,    functional connections cannot be established if the nerve cells no    longer exist. Death of glia, also interferes with nerve function.    The first therapeutic goal is to preserve as many cells as possible,    also known as neuroprotection. Even with neuroprotective drugs or    therapies, some nerve cell death is still likely in SCI. Therefore,    replacement of nerve cells may be required.-   2. Disruption of nerve pathways. The long axons in the ascending and    descending tracts of the spinal cord undergo Wallerian degeneration    after injury. Axonal regeneration must occur to re-establish    neuronal circuits.-   3. Demyelination. Myelin sheaths insulate the long, thin axons to    facilitate nerve impulse transmission. In some types of SCI, as well    as stroke and epilepsy, the nerve cells and axons may not be lost or    interrupted; neuronal dysfunction may be due to loss of the myelin    sheath. This type of damage may be the most amendable to treatment    because rewiring of complex circuits may not be necessary and    remyelination of axons is known to be possible.    Possible Therapies    1. Replacement of Nerve Cells

Mature nerve cells cannot divide to heal a wound. Replacement of lostnerve cells would require transplantation into the site of injury withthe hope that grafted nerve cells would mature and integrate into thehost nervous system. Use of human fetal tissue has shown promise in somestudies, however, it presents ethical and technological considerationsregarding donor tissues and important questions about immune rejectionof transplanted cells. Very recently, scientists have discovered thepresence of adult neural stem cells that can be stimulated to divide anddevelop into neurons and glia. This exciting finding has opened up newpossibilities for cell therapy.

2. Regeneration of Damaged Axons

Neurons in both the central (CNS) and peripheral (PNS) nervous systemsare intimately associated with glia. After injury, CNS glia largelyinhibit regeneration, whilst in the PNS, the Schwann cells facilitateregeneration. The cells are seeded in specially designed “guidancechannels” that have been shown to promote the regeneration of nervefibres in severed rat spinal cords. Schwann cells and neurons producegrowth factors. By introducing these factors into injury sites, alone orin combination with grafts, these have shown that they can stimulateadditional spinal cord regeneration. Schwann cells can be geneticallyengineered to produce growth factors, and these also improveregeneration. Some improvement in hind limb motor function have beenobserved after grafting, however, the results are not reliable enoughyet to justify clinical trials of these procedures. Two exciting newstudies show that olfactory ensheathing glia can “usher” long nervefiber growth into surviving spinal cord regions beyond the site of SCI,after these fibers exit a Schwann cell bridge or grow past the site ofinjury. These promising studies give hope that successful restoration offunction after SCI, stroke or epilepsy may occur one day.

3. Remyelination of Axons

Schwann cells are the cells in peripheral nerves that form myelinsheaths. They are not usually found in the brain or spinal cord whereoligodendrocytes are responsible for myelin production. Researchers haveshown that Schwann cells grafted into the brain can myelinate centralaxons. When the loss of myelin is an important part of an injury,implanting Schwann cells could stimulate remyelination and perhapsrestore function. A multi-center clinical trial has been initiated atother research centers to study a drug (4-AP) that appears to temporallyrestore signal transmission through demyelinated nerve fibers.

CNS Myelin and its Major Inhibitory Effects During Axonal Regeneration

An important barrier to regeneration is the axon growth inhibitoryactivity that is in CNS myelin and that is also associated with theplasma membrane of oligodendrocytes, the cells that synthesize myelin inthe CNS. The growth inhibitory properties of CNS myelin have beendemonstrated in a number of different laboratories by a wide variety oftechniques, including plating neurons on myelin substrates or cryostatsections of white matter, and observations of axon contact with matureoligodendrocytes. Therefore, it is well documented that adult neuronscannot extend neurites over CNS myelin in vitro. It has also been welldocumented that removing myelin in vivo improves the success ofregenerative growth over the native terrain of the CNS. Regenerationoccurs after irradiation of newborn rats, a procedure that killsoligodendrocytes and prevents the appearance of myelin proteins. Aftersuch a procedure in rats and combined with a corticospinal trait lesion,some corticospinal axons regrow long distances beyond the lesions. Also,in a chick model of spinal cord repair, the onset of myelinationcorrelates with a loss of its regenerative ability of cut axons. Theremoval of myelin with anti-galactocerebroside and complement in theembryonic chick spinal cord extends the permissive period for axonalregeneration. Known inhibitory molecules in myelin include myelinassociated glycoprotein (MAG), tenascin-R (TN-R), arretin, andchondroitin-sulphate proteoglycans (CSPGs). Recently, three groupsreported the identification in rats and humans of a gene, Nogo, whichencodes an inhibitory myelin protein (GrandPre et al, 2000; Prinjha etal, 2000; Chen et al, 2000). Immunization against myelin has been foundto allow extensive axon regeneration after injury,—this demonstrates theenormous potential value of overcoming myelin inhibition. Theseexperiments demonstrate a good correlation between inhibitory factors inmyelin and the failure of axons to regenerate in the CNS.

Multiple Sclerosis

MS is a degenerative central nervous system disorder involving decreasednerve function associated with the formation of scars on the insulatingsheath known as myelin around nerve cells.

The cause of MS is not known. However, many researchers believe it maybe an autoimmune disease, perhaps triggered by a viral infection. Thereis no definitive clinical test for the diagnosis of MS. However, an MRI(Magnetic Resonance Imaging) can show areas in the brain where myelinhas been damaged.

MS affects approximately 250,000-300,000 people in the US. Itpredominantly afflicts women, Caucasians, and people from temperateclimates. Generally, the onset of MS is diagnosed in people ages 20-40.

It is now well accepted that MS lesions contain substantial numbers ofpremyelinating oligodendrocytes, indicating that:

-   -   The potential for repair is not limited by the loss of these        cells;    -   Interactions between oligodendrocytes and their surrounding        environment may determine the outcome of the repair process.

FIRST ASPECT OF THE INVENTION Background to First Aspect of theInvention

Nogo-A has been extensively studied in the context of CNS regenerationand is a pivotal factor in the inhibition of axonal regeneration afterinjury (Woolf, 2003). The three major molecules responsible for thegrowth inhibitory property of CNS myelin—Nogo-A, myelin-associatedglycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) appearto bind to the same glycosylphosphatidyl inositol (GPI)-anchored Nogo-66receptor (NgR) on the surface of axons (Woolf, 2003). Understanding ofthe molecular interactions involved in inhibiting neuronal regenerationhas led to the exciting possibility that the CNS environment can bemanipulated to enhance neuronal regeneration. Apart from these findings,however, other paradigms for Nogo-A action in the CNS have yet to bedetermined. For example, the localization of the presence of Nogo-A atsynapses (Wang et al, 2002) is suggestive of a possible role inmodulating synaptic plasticity.

Nogo is expressed in three isoforms, Nogo-A, B and C, all of which sharethe same C-terminus with two transmembrane domains and an extracellular66 amino acid loop (Nogo-66) (GrandPre et al, 2000; Fournier et al,2001). Nogo-A has a large cytoplasmic N-terminal domain (Nogo-N) notfound in Nogo-B or Nogo-C (GrandPre et al, 2000; Prinjha et al, 2000;Brittis et al, 2001). Nogo-66 and Nogo-N of Nogo-A have independentinhibitory activity (Fournier et al, 2001; GrandPre et al, 2000). TheNogo-66 loop on the oligodendrocyte surface binds to the Nogo-66receptor (NgR), which mediates axonal growth retardation (Fournier etal, 2001). In contrast, no receptor or interacting protein for Nogo-Nhas been recognized so far. Nogo-A is expressed in the brain of theearly embryonic CNS, but there is little or no detectable NgR expressionin early embryonic neurons (Wang et al, 2002). Nogo-A therefore may haveNgR independent functions during embryonic stages. Neuronal regenerationphenotypes in the absence of Nogo-A in knockout mice were notparticularly clear, and the exact role of Nogo-A in the CNS remains tobe further investigated (Woolf, 2003). In the adult, although Nogo-A hasbeen localized to oligodendrocytes and NgR to mature axons, the exactdistribution pattern and relationship between these two molecules alongmyelinated axons have not been defined in detail. Knowledge of Nogo-Aand NgR distribution at the interface between neurons andoligodendrocytes will be important in understanding their roles in CNSdevelopment.

The establishment and maintenance of the molecular architecture ofaxonal domains is critical to ensure rapid saltatory conduction of nerveimpulses (Pedraza et al, 2001). During myelination, there is a complex,yet precise and efficient, process that ensures the clustering ofspecific ion channels and other protein molecules to distinct segmentsalong the axon (Dupree et al, 1999; Rasband et al, 2000; 2001a). TheNodes of Ranvier are enriched in Na⁺ channels whilst K⁺ channels areexcluded from this location and instead occupy juxtaparanodal regions.However, during the early stages of both developmental myelination andremyelination, K⁺ channel clusters are transiently located at theparanodal between the nodes and juxtaparanodes region (Rasband et al,1998; Vabnick et al, 1999). This is where the glial cytoplasmic loopscome into contact with the axolemma. Adhesion molecules, such asF3/Contactin, neurofascin 155, and Caspr (Paranodin), are located at theparanodes (Einheber et al, 1997; Menegoz et al, 1997; Tait et al, 2000;Kazarinova-Noyes et al, 2001). Studies on dysmyelinating mouse mutantsdeficient in myelin-related and axonal proteins, such as ceramidegalactosyl transferase (CGT) (Ishibashi et al, 2002; Popko B, 2000),F3/Contactin (Boyle et al, 2001), or Caspr (Bhat et al, 2001), haveshown that clustering of axonal domain constituents and the exactlocalization of ion channels, particularly K⁺ channels, are dependent oncommunication between axons and oligodendroglia. However, the molecularmechanisms that regulate the accumulation of K⁺ channels into compactzones are not entirely clear.

Contactin-associated protein (Caspr) is a transmembrane protein with anextracellular domain that contains a series of laminin G-like domainsand EGF repeats (Peles et al, 1997), as well as a cytoplasmic segmentwith potential binding sites for SH3-containing proteins and 4.1 familyproteins (Gollan et al, 2002). Caspr exists in a complex withF3/Contactin, the GPI-anchored molecule (Peles et al, 1997). Duringmyelination, the interaction of Caspr with F3/Contactin is required forthe proper transport of Caspr and Na⁺ channels to the cell surface(Faivre-Sarrailh et al, 2000; Kazarinova-Noyes et al, 2001). Moreover,this complex constitutes an essential scaffold to maintain thearchitecture of the axoglial apparatus (Bhat et al, 2001; Boyle et al,2001). Although neurofascin 155 (NF155) interacts in trans with theCaspr/F3 complex (Charles et al, 2002), the functional consequence ofthis interaction is still unclear. Judging from Caspr's multiple domainstructure, there may exist other glial components that interact withthis molecule during myelination.

Summary of the First Aspect of the Invention

The inventor explored whether Nogo-A could have a role in the axoglialjunction during the period of myelination—in particular, if Nogo-Aparticipates in molecular interactions between axons andoligodendrocytes. He has determined that Nogo-A can be found localizedspecifically to paranodes, where it interacts with the paranodaljunction protein, Caspr. The inventor has also determined that bothNogo-A and Caspr associate with the voltage-gated K⁺ channel Kv1.1.Furthermore, the co-localization patterns of Nogo-A/Kv1.1 andCaspr/Kv1.1 during development are closely related. The inventortherefore believes that the interaction of Nogo-A with Caspr plays arole in regulating the location of K⁺ channels along the axon during theearly period of myelination.

Specifically, the work carried out by the inventor has shown thatoligodendrocyte Nogo-A is clustered at specific axoglial junctions,where it interacts directly, via its extracellular Nogo-66 loop withaxonal Caspr, and indirectly with K⁺ channel proteins. This representsthe first NgR-independent Nogo-66 interaction described to date, and hassignificant implications for the role of Nogo-A in the formation andmaintenance of axoglial junction architecture.

Thus, at its most general, the present invention provides materials andmethods arising from the determination that Nogo-A and Caspr interactand play a role in myelination. This has important implicationsparticularly in the field of spinal cord injury or other diseases thatresult in damage to the myelin sheaths.

The invention provides a composition comprising Nogo and Caspr, ormimetics thereof, or a substance capable of promoting interactionbetween Nogo and Caspr, in combination with a suitable carrier.

Preferably the composition comprises a complex between Nogo and Caspr,or a mimetic of said complex.

The Nogo molecule present in the composition is preferably Nogo-A or aportion or domain thereof. Preferably it comprises Nogo-66, which isfound in all three known isoforms of Nogo (A, B and C). It may furthercomprise other domains of those isoforms; alternatively the Nogo-66domain may be present in the absence of further portions of Nogoproteins.

The Caspr molecule of the composition is preferably Caspr1 or a portionor domain thereof.

A substance capable of promoting interaction between Nogo and Caspr maybe of any molecular type, including, but not limited to a protein,peptide, or small molecule.

Typically, the susbstance capable of promoting such interaction willbind to one or both of Nogo and Caspr. For example, it may bind to bothproteins, e.g at the interface between Nogo and Caspr. Alternatively itmay bind to only one of Nogo and Caspr, possibly stabilising theconformation of that protein in the complex and so promoting associationand/or inhibiting dissociation of the complex.

As an example, the substance capable of promoting interaction betweenNogo and Caspr may be an antibody, such as an antibody capable ofbinding to both Nogo and Caspr, e.g. a bispecific antibody.

The composition may be a pharmaceutical composition, in which case thecarrier will be of a pharmaceutically acceptable type. Preferably thepharmaceutical composition is formulated for injection in vivo, morepreferably for injection directly into the CNS. Specifically, there isprovided a pharmaceutical composition comprising Nogo-A and Caspr.

The invention further provides a composition as described above for usein a method of medical treatment, and particularly for use in thetreatment of injury to, or disease of, the CNS, such as spinal cordinjury (SCI), multiple sclerosis (MS), epilepsy or stroke.

The invention further provides the use of Nogo in the preparation of amedicament for the treatment of injury to, or disease of, the CNS,wherein the medicament is for administration in combination with Caspror a mimetic thereof.

Likewise, the invention provides the use of Caspr in the preparation ofa medicament for the treatment of injury to, or disease of, the CNS,wherein the medicament is for administration in combination with Nogo ora mimetic thereof.

Thus the medicament may comprise both Nogo and Caspr or mimetics thereof(i.e. it may be a pharmacaeutical composition as described above). Theinvention accordingly provides a method of manufacturing apharmaceutical compostion comprising admixing Nogo and Caspr or mimeticsthereof, with a pharmaceutically acceptable carrier. Alternatively thetwo components may be administered separately.

Also provided is the use of a substance capable of promoting interactionbetween Nogo and Caspr, as herein described, in the preparation of amedicament for the treatment of injury or disease to the CNS.

The invention also provides a method of stimulating myelination of aneuron, specifically a neural axon, comprising contacting a neuron or anoligodendrocyte with a composition as described above. This may beperformed in vivo, e.g. as a therapeutic method as elsewhere describedin this specification, or in vitro.

Also provided is a method of treating a subject having disease of orinjury to the central nervous system, comprising administering to thesubject one or more pharmaceutical compositions comprising Nogo andCaspr as described above. Specifically, there is provided a method oftreating a patient with disease or injury to the CNS, e.g. SCI, MS,epilepsy or stroke, comprising administering to the patient a complexcomprising Nogo-A and Caspr.

All therapeutic methods described are considered particularlyappropriate for the treatment of spinal cord injury (SCI), multiplesclerosis (MS), epilepsy or stroke.

The invention further provides a method of screening for a substancecapable of modulating (preferably promoting) interaction between Nogoand Caspr, the method comprising contacting Nogo and Caspr with acandidate substance, and determining the interaction between Nogo andCaspr.

The method may further comprise contacting Nogo and Caspr in the absenceof said candidate substance under otherwise analogous conditions, anddetermining the interaction between Nogo and Caspr.

Preferably the method comprises contacting a complex between Nogo andCaspr with the candidate substance; the complex is preferably formedbefore it is contacted with the candidate substance.

The method may be performed by any appropriate method. The skilledperson will be well aware of many suitable assay formats, and will bewell capable of designing a suitable protocol.

One or both of Nogo and Caspr may be present in, or on, a cell. The genefrom which the protein is expressed may be endogenous to the cell inquestion, or it may be present on a vector introduced into the cell. Theprotein is preferably expressed on the surface of the cell.

Additionally or alternatively, one or both of Nogo and Caspr may beimmobilised on a solid support. One or both may comprise a detectablelabel as described in more detail below.

The invention further provides a method of manufacturing apharmaceutical formulation comprising, having identified a substancecapable of modulating interaction between Nogo and Caspr by a screeningmethod described herein, the further step of formulating said substancewith a pharmaceutically acceptable carrier. The method may comprise thefurther step of optimising said identified substance for administrationin vivo prior to formulation.

Cells

The term oligodendrocyte is used herein to refer to oligodendroglialcells capable of laying down a myelin sheath around a neuronal axon inthe central nervous system (CNS).

Protein Sequences

The term “Nogo” is used to encompass all isoforms of the Nogo protein,including Nogo-A, B and C, as well as portions and isolated domainsthereof, including the Nogo-66 domain, as well as mutants and variantsthereof having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to the sequences given below. Orthologous proteins from othermammalian species are also included. Preferably the Nogo protein has theability to bind to a Caspr protein, particularly Caspr-1. Nogo-A isparticularly preferred.

The term “Caspr” is used to encompass all isoforms of the Nogo protein,including Caspr-1,2, 3 and 4. Caspr-1 is particularly preferred. Theterm is also intended to encompass isolated domains of such Casprproteins such as the extracellular domain, as well as mutants andvariants thereof having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% identity to the sequences given below. Orthologous proteins fromother mammalian species are also included. The Caspr protein preferablyhas the ability to bind to a Nogo protein, particularly Nogo-A. It mayalso have the ability to bind to at least one subunit of a voltage-gatedpotassium channel, in particular Kv1.1 and/or Kv1.2.

The amino acid sequences of Nogo and Caspr proteins are shown below,along with their GenBank accession numbers; Nogo-A gi:9408096 (CAB99248)1 medldqsplv sssdspprpq pafkyqfvre pedeeeeeee eeedededle elevlerkpa 61aglsaapvpt apaagaplmd fgndfvppap rgplpaappv aperqpswdp spvsstvpap 121splsaaavsp sklpeddepp arppppppas vspqaepvwt ppapapaapp stpaapkrrg 181ssgsvdetlf alpaasepvi rssaenmdlk eqpgntisag qedfpsvlle taaslpslsp 241lsaasfkehe ylgnlstvlp tegtlqenvs easkevseka ktllidrdlt efseleysem 301gssfsvspka esavivanpr eeiivknkde eeklvsnnil hnqqelptal tklvkedevv 361ssekakdsfn ekrvaveapm reeyadfkpf ervwevkdsk edsdmlaagg kiesnleskv 421dkkcfadsle qtnhekdses snddtsfpst pegikdrpga yitcapfnpa atesiatnif 481pllgdptsen ktdekkieek kaqivteknt stktsnpflv aaqdsetdyv ttdnltkvte 541evvanmpegl tpdlvqeace selnevtgtk iayetkmdlv qtsevmqesl ypaaqlcpsf 601eeseatpspv lpdivmeapl nsavpsagas viqpsssple assvnyesik hepenpppye 661eamsvslkkv sgikeeikep eninaalqet eapyisiacd liketklsae papdfsdyse 721makveqpvpd hselvedssp dsepvdlfsd dsipdvpqkq detvmlvkes ltetsfesmi 781eyenkeklsa lppeggkpyl esfklsldnt kdtllpdevs tlskkekipl qmeelstavy 841snddlfiske aqiretetfs dsspieiide fptlissktd sfsklareyt dlevshksei 901anapdgagsl pctelphdls lkniqpkvee kisfsddfsk ngsatskvll lppdvsalat 961qaeiesivkp kvlvkeaekk lpsdtekedr spsaifsael sktsvvdlly wrdikktgvv 1021fgaslfllls ltvfsivsvt ayialallsv tisfriykgv iqaiqksdeg hpfraylese 1081vaiseelvqk ysnsalghvn ctikelrrlf lvddlvdslk favlmwvfty vgalfngltl 1141lilalislfs vpviyerhqa qidhylglan knvkdamaki qakipglkrk ae Nogo-Bgi:9408098 (CAB99249) 1 medldqsplv sssdspprpq pafkyqfvre pedeeeeeeeeeedededle elevlerkpa 61 aglsaapvpt apaagaplmd fgndfvppap rgplpaappvaperqpswdp spvsstvpap 121 splsaaavsp sklpeddepp arppppppas vspqaepvwtppapapaapp stpaapkrrg 181 ssgsvvvdll ywrdikktgv vfgaslflll sltvfsivsvtayialalls vtisfriykg 241 viqaiqksde ghpfrayles evaiseelvq kysnsalghvnctikelrrl flvddlvdsl 301 kfavlmwvft yvgalfnglt llilalislf svpviyerhqaqidhylgla nknvkdamak 361 iqakipglkr kae Nogo-C gi:9408100 (CAB99250) 1mdgqkknwkd kvvdllywrd ikktgvvfga slflllsltv fsivsvtayi alallsvtis 61friykgviqa iqksdeghpf raylesevai seelvqkysn salghvncti kelrrlflvd 121dlvdslkfav lmwvftyvga lfngltllil alislfsvpv iyerhqaqid hylglanknv 181kdamakiqak ipglkrkae Caspr1 gi:4505463 (NP003623) 1 mmhlrlfcillaavsgaegw gyygcdeelv gplyarslga ssyyslltap rfarlhgisg 61 wsprigdpnpwlqidlmkkh riravatqgs fnswdwvtry mllygdrvds wtpfyqrghn 121 stffgnvnesavvrhdlhfh ftaryirivp lawnprgkig lrlglygcpy kadilyfdgd 181 daisyrfprgvsrslwdvfa fsfkteekdg lllhaegaqg dyvtlelega hlllhmslgs 241 spiqprpghttvsaggvlnd qhwhyvrvdr fgrdvnftld gyvqrfilng dferlnldte 301 mfigglvgaarknlayrhnf rgcienvifn rvniadlavr rhsritfegk vafrcldpvp 361 hpinfggphnfvqvpgfprr grlavsfrfr twdltglllf srlgdglghv eltlsegqvn 421 vsiaqsgrkklqfaagyrln dgfwhevnfv aqenhavisi ddvegaevrv sypllirtgt 481 syffggcpkpasrwdchsnq tafhgcmell kvdgqlvnlt lvegrrlgfy aevlfdtcgi 541 tdrcspnmcehdgrcyqswd dficyceltg ykgetchtpl ykesceayrl sgktsgnfti 601 dpdgsgplkpfvvycdiren rawtvvrhdr lwttrvtgss merpflgaiq ywnasweevs 661 alanasqhceqwiefscyns rllntaggyp ysfwigrnee qhfywggsqp giqrcacgld 721 rscvdpalycncdadqpqwr tdkglltfvd hlpvtqvvig dtnrstseaq fflrplrcyg 781 drnswntisfhtgaalrfpp iranhsldvs fyfrtsapsg vflenmggpy cqwrrpyvrv 841 elntsrdvvfafdvgngden ltvhsddfef nddewhlvra einvkqarlr vdhrpwvlrp 901 mplqtyiwmeydqplyvgsa elkrrpfvgc lramrlngvt lnlegranas egtspnctgh 961 cahprlpcfhggrcverysy ytcdcdltaf dgpycnhdig gffepgtwmr ynlqsalrsa 1021 arefshmlsrpvpgyepgyi pgydtpgyvp gyhgpgyrlp dyprpgrpvp gyrgpvynvt 1081 geevsfsfstssapavllyv ssfvrdymav likddgtlql ryqlgtspyv yqlttrpvtd 1141 gqphsinitrvyrnlfiqvd yfplteqkfs llvdsqldsp kalylgrvme tgvidpeiqr 1201 yntpgfsgclsgvrfnnvap lkthfrtprp mtaelaealr vqgelsesnc gamprlvsev 1261 ppeldpwylppdfpyyhdeg wvaillgflv aflllglvgm lvlfylqnhr ykgsyhtnep 1321 kaaheyhpgskpplptsgpa qvptptaapn qapasapapa ptpapapgpr dqnlpqilee 1381 srse

Nogo-66 extends from amino acids 823 to 888 of Nogo-A and has thefollowing sequence: Nogo-66 RIYKGVIQ AIQKSDEGHP FRAYLESEVA ISEELVQKYSNSALGHVNCT IKELRRLFLV DLVDSLKAssay Methods

As described above, the skilled person is well aware of numerous assayformats which may be appropriate for determining interaction betweenNogo and Caspr, and identifying substances which modulate, preferablypromote, such interaction.

For example, interaction between the two proteins may be studied invitro by labelling one with a detectable label and bringing it intocontact with the other which has been immobilised on a solid support.Suitable detectable labels, especially for petidyl substances, include³⁵S-methionine which may be incorporated into recombinantly producedpeptides and polypeptides. Alternatively the complex formed on the solidsupport may be detected by labelling with an antibody directed againstan epitope present on the protein which is not immobilised on the solidsupport. If no suitable antibody is available, a recombinantly-producedpeptide or polypeptide may be expressed as a fusion protein containingan epitope against which a suitable antibody is available.

The protein which is immobilized on a solid support may be immobilizedusing an antibody against that protein bound to a solid support or viaother technologies which are known per se. A preferred in vitrointeraction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described above atest compound can be assayed by determining its ability to affect theamount of labelled peptide or polypeptide which binds to the immobilizedGST-fusion polypeptide. This may be determined by fractionating theglutathione-agarose beads by SDS-polyacrylamide gel electrophoresis.Alternatively, the beads may be rinsed to remove unbound protein and theamount of protein which has bound can be determined by counting theamount of label present in, for example, a suitable scintillationcounter.

An assay according to the present invention may also take the form of acell-based assay in which at least one of the two proteins is expressedby, preferably on the surface of, a suitable cell. The assay may utilisea cell line, such as a yeast strain or mammalian cell line, in which therelevant polypeptides or peptides are expressed from one or more vectorsintroduced into the cell.

Modulators of Nogo-Caspr interaction identified by the methods describedmay be further modified to increase their suitability for in vivoadministration.

Formulations

The compositions of the invention may be prepared as pharmaceuticalformulations comprising at least one active compound, as defined above,together with one or more other pharmaceutically acceptable ingredientswell known to those skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, adjuvants, excipients, buffers,preservatives and stabilisers. The formulation may further compriseother active agents.

Thus, the present invention further provides a method of making apharmaceutical composition as previously defined, the method comprisingadmixing at least one active agent as decribed herein together with oneor more pharmaceutically acceptable ingredients well known to thoseskilled in the art, e.g., carriers, adjuvants, excipients, etc.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, adjuvant, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

Suitable carriers, adjuvants, excipients, etc. can be found in standardpharmaceutical texts, for example Remington's Pharmaceutical Sciences,20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook ofPharmaceutical Excipients, 2nd edition, 1994.

Formulations may suitably be injectable formulations, e.g. in the formof aqueous, isotonic, pyrogen-free, sterile solutions, in which theactive compound is dissolved. Such liquids may additional contain otherpharmaceutically acceptable ingredients, such as anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, suspending agents, thickeningagents, and solutes which render the formulation isotonic with the bloodor cerebrospinal fluid. Examples of suitable isotonic carriers for usein such formulations include Sodium Chloride Injection, Ringer'sSolution, or Lactated Ringer's Injection. Typically, the concentrationof the active compound in the liquid is from about 1 ng/ml to about 10μg/ml, for example from about 10 ng/ml to about 1 μg/ml. Theformulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in afreeze-dried (lyophilised) condition requiring only the addition of thesterile liquid carrier, for example water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules, and tablets.

Administration

Administration of the compositions of the invention will generally be byinjection, preferably directly into the CNS. Injection may be directlyinto the site of damage. Alternatively, injection may be into thecerebro-spinal fluid, typically near the site of disease or injury.

Sequence Identity

Percent (%) amino acid sequence identity with respect to a referencesequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe reference sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. % identity values may be determined by WU-BLAST-2(Altschul et al., Methods in Enzymology, 266:460-480 (1996)). WU-BLAST-2uses several search parameters, most of which are set to the defaultvalues. The adjustable parameters are set with the following values:overlap span=1, overlap fraction=0.125, word threshold (T)=11. A % aminoacid sequence identity value is determined by the number of matchingidentical residues as determined by WU-BLAST-2, divided by the totalnumber of residues of the reference sequence (gaps introduced byWU-BLAST-2 into the reference sequence to maximize the alignment scorebeing ignored), multiplied by 100.

Percent (%) amino acid similarity is defined in the same way asidentity, with the exception that residues scoring a positive value inthe BLOSUM62 matrix are counted. Thus, residues which are non-identicalbut which have similar properties (e.g. as a result of conservativesubstitutions) are also counted.

In a similar manner, percent (%) nucleic acid sequence identity withrespect to a reference nucleic acid is defined as the percentage ofnucleotide residues in a candidate sequence that are identical with thenucleotide residues in the reference nucleic acid sequence. The identityvalues used herein may be generated by the BLASTN module of WU-BLAST-2set to the default parameters, with overlap span and overlap fractionset to 1 and 0.125, respectively.

The Subject

The subject to which the compositions and/or treatments of the inventionwill be administered will be a mammal, preferably an experimental animalsuch as a rodent (e.g. a rabbit, rat or mouse), dog, cat, monkey or ape,or a farm animal such as a cow, horse, sheep, pig or goat. Morepreferably, the subject is human.

Generally, the subject will have CNS damage, caused by disease orinjury, e.g. a head injury. More preferably, however, the damage is tothe spinal cord, e.g. SCI. In experimental animals, the damage may beexperimental. The CNS damage may also result from a disease or disorder,e.g. stroke, epilepsy or a neurodegenerative condition, learningmemory-related condition and/or dementia such as Alzheimer's disease orParkinson's disease.

The treatments of the invention will generally be intended for use inconjunction with other therapies, such as surgery and/or rehabilitation.

Mimetics

Non-peptide “small molecules” are often preferred to peptides orpolypeptides for in vivo pharmaceutical use. Accordingly, mimetics ofCaspr and/or Nogo may be designed, especially for pharmaceutical use.Typically a Nogo mimetic of the present invention will be capable ofbinding to a Caspr molecule to mimic the effects of Nogo binding to thatmolecule. Likewise a Caspr mimetic will be capable of binding to a Nogomolecule to mimic the effects of Caspr binding to that molecule.

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable where the active compound is difficultor expensive to synthesise or where it is unsuitable for a particularmethod of administration, e.g. peptides are unsuitable active agents fororal compositions as they tend to be quickly degraded by proteases inthe alimentary canal. Mimetic design, synthesis and testing is generallyused to avoid randomly screening large number of molecules for a targetproperty.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g. by substituting each residue in turn. Alanine scans of peptide arecommonly used to refine such peptide motifs. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modelledaccording to its physical properties, eg stereochemistry, bonding, sizeand/or charge, using data from a range of sources, eg spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conveniently be selected so thatthe mimetic is easy to synthesise, is likely to be pharmacologicallyacceptable, and does not degrade in viva, while retaining the biologicalactivity of the lead compound. Alternatively, where the mimetic ispeptide based, further stability can be achieved by cyclising thepeptide, increasing its rigidity. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimisation ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

In the present case, peptide mapping studies may be used to identify theminimal portion of one protein required to interact with the other. Thispeptide may then be used as a lead compound for mimetic design, asdescribed above.

Antibodies

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding substance having anbinding domain with the required specificity. Thus, this term coversantibody fragments, derivatives, functional equivalents and homologuesof antibodies, including any polypeptide comprising an immunoglobulinbinding domain, whether natural or synthetic. Chimaeric moleculescomprising an immunoglobulin binding domain, or equivalent, fused toanother polypeptide are therefore included. Cloning and expression ofchimaeric antibodies are described in EP-A-0120694 and EP-A-0125023.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston etal, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993).

Diabodies are multimers of polypeptides, each polypeptide comprising afirst domain comprising a binding region of an immunoglobulin lightchain and a second domain comprising a binding region of animmunoglobulin heavy chain, the two domains being linked (eg by apeptide linker) but unable to associate with each other to form anantigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804).

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449(1993)), eg prepared chemically or from hybrid hybridomas, or may be anyof the bispecific antibody fragments mentioned above. It may bepreferable to use scFv dimers or diabodies rather than whole antibodies.Diabodies and scFv can be constructed without an Fc region, using onlyvariable domains, potentially reducing the effects of anti-idiotypicreaction. Other forms of bispecific antibodies include the single chain“Janusins” described in Traunecker et al, Embo Journal, 10, 3655-3659,(1991).

It may be desirable to “humanise” non-human (eg murine) antibodies toprovide antibodies having the antigen binding properties of thenon-human antibody, while minimising the immunogenic response of theantibodies, eg when they are used in human therapy. Thus, humanisedantibodies comprise framework regions derived from human immunoglobulins(acceptor antibody) in which residues from one or more complementarydetermining regions (CDR's) are replaced by residues from CDR's of anon-human species (donor antibody) such as mouse, rat or rabbit antibodyhaving the desired properties, eg specificity, affinity or capacity.Some of the framework residues of the human antibody may also bereplaced by corresponding non-human residues, or by residues not presentin either donor or acceptor antibodies. These modifications are made tothe further refine and optimise the properties of the antibody.

Specific embodiments of the first aspect of the present invention willnow be illustrated, by way of example, with reference to theaccompanying figures. Further embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES RELATING TO THE FIRST ASPECT OF THEINVENTION

FIG. 1. Glia-derived Nogo-A clusters at the paranodes in CNS.

A. Adult rat brain stem sections were double immunolabeled for Nogo-A(green; a, c, d, f, g, and i) and the Kv1.1 K⁺ channel α-subunit (red;b, c, h, and i) or PAN Na⁺ channel (red; e and f). For negative control,Nogo-A antiserum (1:200) was premixed with antigen before staining of anadult brain stem section (j). Two Nogo-A antibodies, one developed inthe inventor's lab (a-f) and another was developed in Dr. Stritmatter'slab (g-i), were used in this study. c, f and i are merged images of a-b,d-e and g-h, respectively.

B. Ultrastructural localization of Nogo-A at the paranodes in rat spinalcord. (a) Immunogold labeling of cross sections of myelinated axonsrevealed that the gold particles were detected at the inner and outermyelin sheaths. (b and c) Immunogold particles of Nogo-A are foundwithin glial loops and the compacted myelin. (d and e) In longitudinalsections of paranodes, immunogold particles of Nogo-A located at thetips of glial loops in the axoglial junction and some within the axon ind. A higher magnification of the boxed areas of d and e were shown in d′and e′. ax: axon; OL: oligodendrocyte. Gold particles are indicated witharrows. Bars: 5 μm for Aa-i, 10 μm for Aj, 200 nm for Ba, d and e, 100nm for Bb, c, d′ and e′.

FIG. 2. Expression and localization of Nogo-A in the CNS myelinatedaxons from EAE rats and CGT ^(−/−) mice. A and B. Spinal cord sectionsfrom EAE rats were double immunostained for Nogo-A (green, A), Caspr(green, B) and Kv1.1 (red, A and B). The star marks a Nogo-A positivecell body, and the arrowhead indicates an undisrupted paranodal Nogo-Alabeling. C. Numbers of Nogo-A and Caspr clusters in several microscopicfields of both normal and EAE rat spinal cord sections were counted.Values are given as mean±SEM from at least 3 independent experiments.Nogo-A clusters were dramatically reduced in EAE compared to normalrats. The double asterisks (**) indicates a significance level ofP<0.01. D. Western blotting result showing that Nogo-A was significantlydownregulated in spinal cord of EAE animals, but Caspr expression wasonly affected slightly. The single asterisk represents a significancelevel of P<0.05. E, F, G, and H. Spinal cord sections from wild type andCGT ^(−/−) mice were double-labeled for Nogo-A (green) and PAN Na⁺channel. In wild-type (P16), Nogo-A clusters at paranodes and PAN Na⁺channels congregated at the nodes of Ranvier (E). In CGT^(−/−) mice(P16), Nogo-A segregation at paranodes and PAN Na⁺ channel clusteringwere hardly detected (F). Nogo-A labeling appears to be loosely spiraledaround the axon in CGT^(−/−) mice (P21; H), but clusters specificallyinto the paranodal region in wild type mice (P21; G). Bars: 10 μm forA-B and 5 μm for E-H. I and J. Immunogold labeling of Nogo-A inlongitudinal sections of paranodes from P16 CGT^(−/−) mice spinal corddemonstrated that gold particles were visible in the abnormally reversedloops. Panels I′ and J′ are high magnification of the boxed areas in Iand J, respectively. Arrows indicate the gold particles. Stars indicatethe reversed papanodal loops. ax: axon. OL: oligodendrocyte. Bars: 200nm for I and J, 50 nm for I′ and J′.

FIG. 3. Caspr associates with Nogo-A in vitro.

A. NgR distributes diffusely along the myelinated axon. (a) Tissuelysates from various regions of the CNS of adult rats were subjected toWestern blot using antibodies against NgR and γ-tubulin. (b) Brainstemsfrom postnatal day 1-30 (P1-P30) rats were subjected to Western blotusing antibodies against NgR and γ-tubulin. Adult hippocampus (c-e) andbrainstem (f-h) sections were double stained for NgR (green, c and f),MAP2 (red, d) and Kv1.1 (red, g). e and h represent merged images of c,d, and f, g, respectively. Scale bars for c to h: 10 μm.

B. Nogo-A associates with Caspr/F3. (a) Detergent lysates of brainmembrane fractions from adult mice were immunoprecipitated with Caspr,Nogo-A, and NB3 antibodies as well as non-immune IgG. Theimmunoprecipitates and detergent extracts from brain (Brain) togetherwith the protein-A beads (Beads) were subjected to Western blot usingantibodies against Caspr, Nogo-A, F3, and NB3. (b) Membrane fractions ofNogo-A/Caspr/F3-, Nogo-A/F3- and Nogo-A-transfected CHO cells wereimmunoprecipitated with antibodies to Caspr and Nogo-A as well asnon-immune IgG. The immunoprecipitates and brain extracts (Brain) weresubjected to Western blot analysis using antibodies against Nogo-A,Caspr and F3. (c) Detergent lysates of membrane fractions from P15 ratcerebral cortex were loaded onto a linear sucrose gradient. Twelvegradient fractions were collected, subjected to SDS-PAGE and analyzedfor the distribution of Nogo-A, Caspr and F3 by Western blotting.Fraction 1 is the lowest density fraction recovered from the top ofgradient. The last lane that is labeled “total” indicates the levels inhomogenates before loading onto the sucrose gradient.

FIG. 4. Caspr expressing cells adhere to Nogo-66.

A to L. Caspr/F3-(A, E and I), F3-(B, F and J), and mock-(C, G and K),as well as PI-PLC treated Caspr/F3-(D, H and L) CHO cells were platedonto substrates coated with Nogo-66 peptide, recombinant GST-Nogo-66protein, and GST, respectively. Scale bar: 8 μm. M to N. Quantificationof cells adherence to various substrates. Caspr/F3-CHO cells (in thepresence or absence of PI-PLC treatment), but neither F3-nor wild typeCHO cells, bound to Nogo-66 peptide and GST-Nogo-66. Bars represent thenumber of adherent cells (expressed as mean±SEM) from at least 3independent experiments (M). O. At the end of cell adhesion assay afterPI-PLC treatment, Caspr/F3-CHO and F3-CHO cells and their culturesupernatant were collected, and subjected to Western blotting analysisafter normalizing for total protein to detect F3/Contactin. 1, mousebrain; 2, F3-CHO cell lysate; 3, Caspr/F3-CHO cell lysate after PI-PLCtreatment; 4, Caspr/F3-CHO cell medium after PI-PLC treatment. Theasterisk (*) indicate a significance level of P<0.05. Caspr/F3-CHO cellsadhered to Nogo-66 and GST-Nogo-66 with equal efficiency after treatmentwith increasing concentrations of PI-PLC (0.02, 0.04 and 0.06 U/ml) (N).

FIG. 5. The Nogo-A/Caspr complex interacts with K⁺ channels.

A. (a) P7 mouse brain membrane extracts were immunoprecipitated withCaspr, Nogo-A, Kv1.1, Kv1.2 and NB3 antibodies as well as non-immuneIgG. The indicated immunoprecipitates and brain extracts (Brain) weresubjected to Western blot analysis using antibodies against Kv1.1,Kv1.2, Nogo-A and Caspr. (b) GST pull-down assay was performed usingrecombinant GST-Nogo-66, GST-Nogo-N and GST from adult mouse brainextracts. The indicated precipitates and brain extracts (brain) wereprobed with Caspr and Kv1.1 antibodies, following SDS-PAGE separation.

B. (a) Membrane fraction of CHO, F3-CHO, Caspr/F3 CHO cells, as well asbrain extracts were immunoblotted using Caspr antibodies, followingSDS-PAGE separation. (b) After transient transfection with the Kv1.1expression construct RBG4/Kv1.1, membrane extracts of CHO, F3-CHO,Caspr/F3 CHO cells were incubated with GST-Nogo-66 or GST, respectively.The eluted proteins were separated by SDS-PAGE and probed with Caspr andKv1.1 antibodies. IP: immunoprecipitation; WB: Western blot.

FIG. 6. Immunohistochemical labeling of Nogo-A, Caspr and Kv1.1 atdifferent postnatal days in rat brainstem.

A. Brainstem sections of P5 to adult rats were double labeled for Caspr(green) and Kv1.1 (red). Scale bar: 5 μm. B. Sections from P1 to P30rats were double labeled for Nogo-A (green) and Kv1.1(red). Scale bar(in o): 5 μm. C. (a) The lengths of Nogo-A, Kv1.1 immunostaining andtheir overlap were measured from micrographs (μm, mean±SEM). (b) Thenumber of overlapping Nogo-A/Kv1.1 or Caspr/Kv1.1 clusters at paranodeswas counted.

FIG. 7. Distribution of Nogo-A, Caspr and Kv1.1 in EAE rats and Shiverermice.

A and B. Double immunofluorescence labeling for Caspr (green) and Kv1.1(red) in brainstem sections of EAE (A) and control (B) rats. The insetsrepresent magnified views of the Kv1.1 labeling. Scale bars: 10 μm forA-B and 5 μm for insets of A-B. C to G. Double labeling for Nogo-A(green) and Kv1.1 (red) in spinal cord sections of wild type (C) andShiverer (D-G) mice. F is a merged picture of D and E. Arrows in C, Fand G indicate Nogo-A positive cell bodies. Bars: 10 μm. H and I. Doubleimmunofluorescence staining of Caspr (green) and Kv1.1 (red) inbrainstem sections of wild-type (H) and Shiverer (I) mice. Bar: 10 μm.J. The distances between paired Kv1.1 staining in EAE with control ratsand Shiverer (Shi) with wild-type (WT) mice were measured frommicrographs (μm, mean±SEM), respectively. The values in EAE rats orShiverer mice were significantly reduced when compared to theircontrols, respectively (asterisks ** indicates a significance level ofP<0.01).

FIG. 8. The interaction between Nogo-A and Caspr at the paranodes mayplay a role during myelination.

In addition to NF155, the inventors' findings suggest that paranodalNogo-A is a glial ligand for neuronally expressed Caspr. ParanodalNogo-A trans-interacts with axonal Caspr, and may play a role in K⁺channel localization during the early stages of myelination (from P5).With the firm establishment of axoglial junctions in the adults, K⁺channels were excluded from paranodes where Nogo-A/Caspr interaction ismaintained, however, the detail mechanism for this separation remains tobe explored. N: Node of Ranvier, PN: paranode, JPN: juxtaparanode.

DETAILED DESCRIPTION OF THE FIRST ASPECT OF THE INVENTION

Results

Nogo-A is Localized to the Paranodes of Myelinated Axons

The distribution of Nogo-A was examined along the white matter tracts ofadult rat brainstem. In longitudinal sections, similar localizationpatterns of Nogo-A were observed with two different Nogo-A antibodies:one was developed in the inventor's lab (FIG. 1A, a-f; Liu et al, 2002)and another was a kind gift from Dr. Stephen Stritmatter (YaleUniversity) (FIG. 1A, g-i; Wang et al, 2002). Nogo-A immunoreactivity(green) was confined specifically to paranodal segments along myelinatedaxons (FIG. 1A), as evidenced by double immunofluorescence labeling withKv1.1 (red; FIG. 1A, a-c, g-i) or the Na⁺ channel (red; FIG. 1A, d-f).The specific labeling of Nogo-A in axonal domains was undetectable afterthe Nogo-A antisera (1:200) were premixed with 100 fold molar excess ofantigen (FIG. 1Aj). The Nogo-A staining (green) flanks nodal Na⁺ channellabeling (red), and is flanked by juxtaparanodal Kv1.1 labeling (red),thus reflecting its paranodal location. Similar observations were madein other nerve fiber-rich CNS sites such as the corpus callosum and thespinal cord (not shown). In the sections examined, Nogo-A was alsodetected in both neurons and oligodendrocytes (not shown), consistentwith other published studies (Huber et al, 2002; Wang et al, 2002; Liuet al, 2002). These observations suggest that Nogo-A may be enriched atthe paranode and is a component of the paranodal protein complex.

The specific paranodal location of Nogo-A was further investigated usingimmuno-electromicroscopy (IEM). Consistent with previous observations(Huber et al, 2002), Nogo-A immuno-reactivity was high in the inner andouter loops of the myelin sheath (FIG. 1Ba), and low in the compactmyelin of rat spinal cord (FIG. 1Bb). Notably, in longitudinal sections,Nogo-A immunoreactivity was high in the expanded terminal glial loops(FIG. 1Bb and c) and the axoglial junction between the loops and theaxolemma (FIG. 1Bd and e) at paranodes, and is present only occasionallyin the paranodal axon (FIG. 1Bd). These observations indicate thatNogo-A is a component of the CNS paranodes.

Nogo-A is a Paranodal Element Mainly Derived from Oligodendroglia

Nogo-A is predominantly expressed in oligodendroglial cell bodies andwhite matter of the adult CNS (Huber et al., 2002). To characterize thecellular origin of the paranodal Nogo-A, we examined Nogo-A'sdistribution in two animal models: experimental autoimmuneencephalomyelitis (EAE), a condition involving progressive CNSdemyelination (Swanborg 2001), and CGT^(−/−) mice known to display apresence of reversed lateral loops but an absence of transverse bands,and abnormal localization of K⁺ channels along their axons (Dupree etal, 1999). At the peak of demyelination in adult EAE rats, theirlongitudinal spinal cord sections were prepared for doubleimmunofluorescence staining for Nogo-A or Caspr, and Kv1.1. The densityof Nogo-A positive paranodal congregates was significantly decreased (byaround 90%) in sections from EAE rats (FIGS. 2A and C) compared to thosefrom control animals. Only occasional foci of Nogo-A positive paranodalclusters (arrowhead; FIG. 2A) and oligodendroglial immunoreactivityremained (star; FIG. 2A). In correlation with the loss of paranodalstaining, Nogo-A expression in the spinal cord was downregulated in EAErats (FIG. 2D). However, Caspr expression was affected to a much lesserextent by this disorder in the spinal cord (FIG. 2B-D).

In P16 wild-type mice, Nogo-A (green) clusters beside the congregatedNa⁺ channels (red) at paranodes (FIG. 2E). In P16 CGT^(−/−) mice,however, congregation of both Nogo-A (green) and Na⁺ channels (red) washardly detected along the axons (FIG. 2F). Immunofluorescence analysison spinal cord sections from P21 wild type and CGT^(−/−) mice wasperformed using another axonal marker, the 200 kDa neurofilament, incombination with Nogo-A antibodies. Loose spiral-like labeling of Nogo-Awas detected along the neurofilament labeled axon in CGT^(−/−) mice(FIG. 2H). This rather deranged labeling pattern is clearly differentfrom the compact clustering pattern of Nogo-A labeling in wild typeanimals (FIGS. 2E and G), revealing that the congregation of the glialNogo-A along the axon was severely disrupted in the mutants. IEMobservations showed that Nogo-A immunoreactivity was clearly present inthe reversed lateral loops at paranodes in these mutant mice (FIGS. 2Iand J). Given widespread demyelination and oligodendroglial damage inEAE rats and abnormal nodal and paranodal structures in CGT^(−/−) mice,lost of Nogo-A congregation at the paranodes suggests that paranodalNogo-A is predominantly associated with OLs.

NgR is Uniformly Distributed Along Myelinated Axons

The inventor next investigated the expression and distribution of theNogo-66 receptor (NgR), to find out if it also exhibits domain specificcongregation patterns in myelinated axons.

Immunoblot analyses using NgR antibodies demonstrated that NgRexpression is higher in adult brainstem, hippocampus, and cerebralcortex, but is much lower in spinal cord (FIG. 3Aa). NgR is detectableas early as postnatal day 1, with its expression level maintained tillpostnatal day 14, and subsequently showing a gradual decrease from 3weeks of age (FIG. 3Ab). To confirm that the NgR antibodies used couldlabel NgR on neurons, the inventor stained rat hippocampal sections andshowed that NgR co-localized with the neuron-specificmicrotubule-associated protein 2 (MAP2) in cell bodies and processes(FIG. 3A, c-e). Double labeling for NgR and Nogo-A is difficult due tothe rabbit polyclonal origin of both antibodies. The inventor insteadperformed double labeling of NgR and Kv1.1. In longitudinal brainstemsections, NgR is uniformly distributed along axons, contrasting with thecongregated K⁺ channel labeling (FIG. 3A, f-h). Similar NgR labeling wasobserved in P1, P5, P14, and P30 age groups (not shown). The observationthat the NgR localization pattern is distinctly different from thecongregated Nogo-A at the paranode raises the possibility that Nogo-Amay interact with an axonal receptor other than NgR in these specificaxon-glial domains.

Nogo-A Interacts with Paranodal Caspr/F3 Complex

Given the paranodal location of Nogo-A, the inventor investigatedwhether paranodal axonal components, such as Caspr, F3, and NB3 (anF3-related molecule) (Lee et al, 2000), are Nogo-A binding partners.Nogo-A, Caspr, F3, and NB3 were immunoprecipitated from membraneextracts of adult rat brain. Western blot analysis of theimmunoprecipitates resolved on SDS-PAGE revealed that Nogo-A, Caspr, andF3, but not NB3, were present in the immunocomplexes pulled down byeither Nogo-A or Caspr antibodies (FIG. 3Ba). Immunoprecipitation (IP)studies were also performed on Caspr/F3-, F3-, and wild type CHO cellstransiently transfected with a Nogo-A expression construct. Transfectionwas performed using Caspr/F3 expressing cells because F3 is required forproper cell surface expression of transfected Caspr (Faivre-Sarrailh etal, 2000). Western blotting demonstrated that Nogo-A and Caspr indeedassociate with each other in Caspr/F3-, but not F3- and wild type CHOcells (FIG. 3Bb). These observations suggest that Nogo-A interactsspecifically with Caspr rather than F3.

The Majority of Nogo-A and Caspr/F3 Complex are not Co-Localized inLipid Rafts

F3 and Caspr are associated with lipid rafts in neurons, transfected CHOcells, and OLs (Buttiglione et al, 1998; Kramer et al, 1999;Faivre-Sarrailh et al, 2000). To investigate the relationship betweenNogo-A and the Caspr/F3 associated microdomains during development, ratcerebral cortexes were lysed and fractionated in a sucrose densitygradient according to an established procedure (Krämer et al, 1999). Themajority of the known raft-associated protein F3 was detected infraction 5, and all Caspr in the lysate was virtually found in thisfraction, suggesting that both molecules are located in neuronal rafts.On the other hand, the majority of Nogo-A was found in fractions 8-12that are enriched in cytoskeleton-associated proteins, although a minorportion was present in fractions 5-7 (FIG. 3Bc). Similar results wereobtained using cerebral cortex lysates of both P15 and adult (notshown). These observations demonstrate that the majority of Nogo-A isnot present in the neuronal rafts in vivo, and provides indirect supportfor the notion that paranodal Nogo-A is derived from theoligodendroglial membrane.

Nogo-A Interacts Directly in Trans with Caspr Via the ExtracellularNogo-66 Loop

The extracellular domain of Nogo-A consists of a 66 amino acid loopbetween its two transmembrane domains, known as the Nogo-66 domain(Fournier et al, 2001). Any interaction in trans between Nogo-A andCaspr should involve the Nogo-66 domain. The inventor next investigatedwhether the Nogo-A and Caspr associate directly in a trans-manner thatis independent of F3 using a cell adhesion assay. Different CHO celllines (expressing Caspr or otherwise) were plated onto substrates coatedwith the Nogo-66 peptide or recombinant GST fusion proteins containingNogo-66 (GST-Nogo-66) or the cytoplasmic N-terminal domain of Nogo-A(GST-Nogo-N) as well as GST. Only Caspr/F3-expressing, but neitherF3-expressing nor wild type CHO cells, adhered readily to Nogo-66 (FIG.4A-C) or GST-Nogo-66 (FIG. 4E-G). None of these CHO cell types adheredwell to GST (FIG. 4I-K) or GST-Nogo-N (not shown). Quantification ofadhering cells indicated that the number of Caspr/F3-CHO cells bindingto both Nogo-66 and GST-Nogo-66 was much higher than in otherexperimental groups (FIG. 4M).

To investigate if Nogo-66 interacts with Caspr only or a binding pocketgenerated by the cell surface Caspr/F3 complex, the inventor removed theGPI-linked F3 using phosphatidylinositol-specific phospholipase C(PI-PLC). After a PI-PLC treatment, Caspr/F3-CHO cells still adhered toboth Nogo-66 and GST-Nogo-66, but not to GST coated substrates (FIG. 4D,H, L and M). To ensure that F3 was completely removed from the cellsurface after PI-PLC treatment, increasing concentrations of PI-PLC(0.02, 0.04 and 0.06 units/ml) were used. Cell binding was constant inall three different concentrations of PI-PLC (FIG. 4N). In agreementwith previous work (Faivre-Sarrailh et al, 2000), Western blot (FIG. 4O)and immunostaining (not shown) using F3 antibodies demonstrated that asignificant amount of F3 was indeed removed from Caspr/F3-CHO cellsafter treatment with PI-PLC. This lack of effect of PI-PLC on the celladherence to Nogo-66 substrates suggests that F3 is not directlyinvolved in the trans-interaction between Nogo-66 and Caspr.

The Nogo-A/Caspr Complex Interacts with K⁺ Channels

During myelination, the proper segregation of K⁺ channels tojuxtaparanodes requires an intact paranodal axoglial junction (Vabnickand Shrager, 1998). This structure is formed and maintained by axonalmolecules such as Caspr and F3, as well as by glial specific moleculesduring myelination and remyelination (Girault and Peles, 2002). Theinventor hypothesized that the interaction of Nogo-A with Caspr forms anaxo-glial signaling connection that could influence K⁺ channel's finallocation at juxtaparanodes during the early stages of myelination.Immunoprecipitation analyses were performed to investigate whether K⁺channels could physically interact with Nogo-A/Caspr complex in the CNS.Both Nogo-A and Caspr antibodies reciprocally precipitated Kv1.1 andKv1.2, but not Kv2.1 (not shown), from P7 and adult (not shown) mousebrain extracts, while NB3 antibody and non-immune IgG did not (FIG.5Aa). Kv2.1 is generally found in neuronal cell bodies and proximaldendrites, but is excluded from axons (Trimmer et al, 1991). In GSTpull-down assays using membrane extracts of adult mouse brain, bothCaspr and Kv1.1 could be pulled down by GST-Nogo-66, but not byGST-Nogo-N or GST itself (FIG. SAb). These results support the notionthat Nogo-66 is a trans-interacting partner of Caspr, and that K⁺channels may interact with the complex.

Nogo-66 Via Caspr Interacts Indirectly with K⁺ Channels

The inventor next investigated whether Nogo-66 could directly interactwith K⁺ channels. Kv1.1 cDNA was transiently transfected into Caspr/F3-,F3- and wild type CHO cells and the membrane extracts were subjected toa GST pull-down assay using GST and GST-Nogo-66 fusion proteins,respectively. Wild type and F3-expressing CHO cells did not expressCaspr (FIG. 5Ba). Western blot analysis showed that both Kv1.1 and Casprcould only be precipitated by GST-Nogo-66, but not GST, from theCaspr/F3-expressing, but not F3-expressing and wild type CHO cells (FIG.5Bb). These results demonstrate that Nogo-66 could interact indirectly,at least in vitro, with K⁺ channels via Caspr.

Nogo-A and Caspr Share a Similar Spatial and Temporal Relationship toKv1.1 Along Myelinated Axons

In view of the potential interaction between the paranodal Nogo-A/Casprand Kv1.1 established above, the inventor explored the dynamicrelationship between Nogo-A/Caspr and Kv1.1 distribution alongmyelinated axons during development. Double immunofluorescence labelingsof Caspr and Kv1.1, and of Nogo-A and Kv1.1, were performed on brainstemsections of rats at various postnatal days. Congregations of both Casprand Kv1.1 labeling were apparent from approximately P5 onwards (FIG.6Aa). From P5-P14 (FIG. 6A, a-c), Caspr staining at paranodes overlappedthat of Kv1.1, suggesting co-localization of both molecules at thiscritical early period of myelination. At P30 (FIG. 6Ad), Kv1.1 labelingbecame more distinctly juxtaparanodal, with only minimal bands ofoverlap with Caspr at paranodal-juxtaparanodal borders. In the adults(FIG. 6Ae), Caspr and Kv1.1 were segregated into their differentmicrodomains along the myelinated axons. Double immunofluorescencestaining for Nogo-A and Kv1.1 at P1 (FIG. 6B, a-c) revealed that Nogo-Awas diffusely labeled along the nerve fibers. At P5 (FIG. 6B, d-f),clustering and aggregation of Nogo-A staining became more evident.However, the staining pattern still did not have well-defined domains orborders. Nodal gaps were apparent and hemi-nodes were seen as well. FromP7 (FIG. 6B, g-i), Nogo-A distribution demonstrated an obviousclustering towards the paranodes. From P5-P14 (FIG. 6B, j-l), there werevarying degree of overlap between congregates of Nogo-A and Kv1.1immunostaining at both paranodal and juxtaparanodal regions. At P30(FIG. 6B, m-o), the Kv1.1 congregates were exclusively localized tojuxtaparanodes, akin to the situation in adult animals.

Co-localization of the Nogo-A/Kv1.1 and Caspr/Kv1.1 was quantified bymeasuring the lengths of Nogo-A and Kv1.1 labeled regions on capturedimages (FIG. 6Ca). The average length of a Nogo-A labeled region wasabout 9 μm at P5, 5 μm at P7, but this shortened to about 2 μm from P14to adult, suggesting that Nogo-A is progressively congregated intonarrower bands during the early stages of myelination. The averagelength of a Kv1.1 labeled region did not demonstrate such a markedchange with time: 6 μm at P5 to 8 μm in the adult. Of note was thechange in terms of the length of overlap between Nogo-A and Kv1.1labeling: it decreased from 4 μm at P5, 2 μm at P14, and 1 μm at P30 toapproximately 0 μm in the adult. This change demonstrates a transientco-localization of Nogo-A and Kv1.1 in paranodal regions before compactmyelin is fully laid down. The degree of co-localization betweenNogo-A/Kv1.1 and Caspr/Kv1.1 was also compared (FIG. 6Cb). FromP5-P14, >60% of paranodes in every field of view were double labeled forNogo-A/Kv1.1 and Caspr/Kv1.1, respectively. In adults, Nogo-A and Casprseparated from Kv1.1 and a complete segregation was observed. Given thatK⁺ channels bind to the Nogo-A/Caspr complex, these observations implythat the Nogo-A/Caspr complex may transiently interact with K⁺ channels,and as such may cooperatively regulate the paranodal localization ofKv1.1 during the early stages of myelination.

Nogo-A and K⁺ Channel in Demyelinating Animal Models

Shiverer is a hypomyelinating mutant mouse that lacks myelin basicprotein (MBP) and has axons with normal oligodendroglial ensheathment,but displays aberrant axoglial junctions and abnormal localization of K⁺channels along its axons (Rasband and Trimmer, 2001a). To investigatethe roles of glia-related molecules in the regulation of K⁺ channellocalization in myelinated axons, the distribution of Nogo-A, Caspr, andKv1.1 was examined in both EAE rats and Shiverer mice. Doubleimmunofluorescence staining demonstrated that both disorganized Caspr(green) and Kv1.1 (red) labelings were still detectable in the paranodalregion of EAE rat (FIG. 7A), compared to normal rat (FIG. 7B) brainstem.In contrast to the location of paranodal Nogo-A (green) andjuxtaparanodal Kv1.1 (red) in the spinal cord sections of normal mice(FIG. 7C), Nogo-A staining was diffused along the axons and itscongregates were hardly detectable in the paranodal region of Shiverermice (FIG. 7D-G). Nogo-A immunoreactivity in OLs, however, remainedintact and distinct (arrows; FIG. 7C, F and G). In accordance withprevious observations (Poliak et al, 2001), disorganized Caspr and Kv1.1labeling co-localized at paranodes in Shiverer mice (FIG. 71) but notnormal mice (FIG. 7H). Quantitative analysis of the distance betweenpaired Kv1.1 immuno-stainings demonstrated that the distances betweenthe pairs were significantly reduced in both EAE and Shiverer miceversus normal animals (P<0.01; FIG. 7J). These observations suggestthat, in both pathological conditions of EAE rats and Shiverer micedisplaying paranodal junction defects, K⁺ channels are actuallyrelocated to the paranodes. Concomitantly, the congregation of Nogo-A atthe paranode was markedly reduced. Thus, in addition to axonalmolecules, certain glia-derived molecules involved in formation ofaxoglial junctions may also be essential for proper K⁺ channellocalization at juxtaparanodes in normal adult animals. At the moment,the molecular structural basis for these changes is unknown to theinventor, but may well be related to the interaction between K⁺ channelsand the Nogo-A/Caspr complex.

Discussion Relating to the First Aspect of the Invention

Nogo-A, but not the Nogo-66 Receptor, is a Hallmark of the Paranode

The location of Nogo-A in oligodendroglia and CNS myelin has alreadybeen described (Huber et al, 2002; Liu et al, 2002; Wang et al, 2002).Specifically, Nogo-A has been localized to oligodendrocyte cell bodiesand processes and to the innermost loop and outer loop of the myelinsheath (Huber et al, 2002). In the developing cerebellum, the timecourse of appearance of Nogo-A mRNA and protein parallel the time framefor myelination, occurring in a period just prior to the expression ofmyelin basic protein. These observations suggest a role for Nogo-Aduring myelination.

Increasing evidence points to the importance of axon-glial communicationin the regulation of oligodendrocyte differentiation (Barres et al,1999; Marcus et al, 2000) and ion channel clustering on neurites (Dupreeet al, 1999; Ishibashi et al, 2002). The inventor has investigatedwhether Nogo-A was localized to distinct sites involved in suchintercellular signaling and has shown that Nogo-A is mainly localized atthe gaps between Na⁺ and K⁺ channels along axons and itsimmunoreactivity is clearly located at sites where glial loops makecontact with the axonal membrane surface in adult CNS. This suggeststhat Nogo-A is a paranodal component, which is further confirmed by theobservations in several pathological animal models. In accordance withNogo-A downregulation, its congregates are significantly reduced atparanodes in EAE animals. Nogo-A immunoactivity presents in the reversedparanodal loops in CGT^(−/−) mice, however, congregated Nogo-A is alsohardly detectable at paranodes in both CGT^(−/−) and Shiverer mice.Altogether, these observations support the notion that Nogo-A is aparanodal glial component (FIG. 8).

The staining pattern and oligodendroglial origin of paranodal Nogo-Araised the question as to whether it interacts with components on theaxonal surface. So far, the only known high affinity neuronal receptorfor Nogo-A is the Nogo-66 receptor (NgR). Previous work has shown thatNgR expression in neurons and along myelinated axons is predominantlyfound in adult animals and that its expression during myelination isminimal (Fournier et al, 2001; Hunt et al, 2002; Wang et al, 2002). Theinventor's results are consistent with these findings, in that they showthat NgR is located in the brainstem on neuronal cell bodies anduniformly distributed along myelinated axons from the early stages ofdevelopment until adulthood. It is intriguing that there is significantNogo-A clustering at paranodes, while the NgR distribution patternremained diffuse along axons. As the congregation of Nogo-A coincideswith the developmental period of myelination, paranodal Nogo-A maytherefore participate in this process and may interact with a moleculeother than NgR, for a function distinct from inhibition of axonalsprouting.

Paranodal Nogo-A is a Glial Ligand of Caspr

At the paranodes, the GPI-anchored axonal F3/contactin exists as acomplex with the membrane protein Caspr. The Caspr/F3 complex interactswith NF155, a glial-derived molecule, in trans, and is an example ofaxoglial molecular connection at the paranode (Girault and Peles, 2002).The inventor has shown that Nogo-A associates specifically with theCaspr/F3 complex, but not NB3, another paranodal molecule, in co-IPassays, implicating an interaction between Nogo-A and the complex invivo. The observation that the majority of CNS Nogo-A is notco-localized with the complex in neuronal lipid rafts implies that thisinteraction probably occurs in a trans-manner, with Nogo-A from the OLmembrane interacting with Caspr/F3 from the axonal membrane. Although itremains a possibility that axonal Nogo-A is a cis-binding partner ofCaspr/F3 complex, and is dependent upon myelination for congregation,this seems unlikely since the paranodal Nogo-A is predominantlyexpressed by OLs in the CNS (Huber et al, 2002: Liu et al, 2002; Wang etal, 2002). To support the notion, the inventor has further shown thatCaspr/F3-expressing CHO cells bind to both substrates coated withNogo-66 peptides and GST-Nogo-66, where the binding must occur in atrans manner. That binding occurs even after removal of F3 from thecells via PI-PLC treatment further implies that Nogo-A interactsdirectly with Caspr (FIG. 8).

Nogo-A May Complement Caspr in Regulating Kv1.1 Location

There is strong evidence suggesting that K⁺ channel accumulation at thejuxtaparanode is influenced by myelinating OLs (Vabnick and Shrager,1998). Shaker-type K⁺ channels are multi-protein complexes composed ofKv1.1, Kv1.2 and Kvβ2 subunits (Rasband and Trimmer, 2001b). In themyelinated axons of the CNS, K⁺ channel labeling becomes more prominentduring the progression of postnatal development, initially localizing tojuxtaparanodes and also to paranodal bands that alternate with Casprimmunoreactivity (Rasband et al, 1999). At later stages of postnataldevelopment, K⁺ channels are excluded from the paranodes and becomeexclusively juxtaparanodal. The inventor has shown that Nogo-A, viaCaspr, associates indirectly with Kv1.1. However, the developmentalchanges in the distribution pattern of Nogo-A and Kv1.1, as well as inthose of Caspr and Kv1.1, are similar, implying that the interactionbetween Kv1.1 and Nogo-A/Caspr complex occurs, at least transiently,when Nogo-A/Caspr colocalizes with Kv1.1 at paranodes during the earlystages of myelination. Nogo-A may therefore play a complementary orregulatory role to Caspr in the organization of mature axonal domainsand in so doing aid in the co-ordinated localization of K⁺ channels tojuxtaparanodes (FIG. 8).

Investigation of Nogo-A (or Nogo-A/Caspr) conditional transgenic orknockout animals in vivo, possibly using OL specific promoters, may helpto further reveal the role of Nogo-A during myelination. According tothe inventor's quantitative analyses, the distances of the gap withinpaired K⁺ channel clusters were significantly reduced. This occurs inconjunction with a significant reduction in Nogo-A clusters in both EAErats and Shiverer demyelinated axons compared to normal myelinatedaxons. These observations imply that K⁺ channels co-localize with Caspragain in the pathological conditions, although it would be interestingto explore whether this transient interaction also occurs duringremyelination.

A point that also warrants further investigation is the structural andfunctional nature of the interaction between Nogo-A, Caspr, and the K⁺channels. It should be noted that the localization of Caspr familymembers demarcate distinct domains in myelinated axons. Caspr2, which isabout 45% identical to Caspr, is localized to the juxtaparanodes ofadult myelinated axons. It associates with K⁺ channels indirectly viaits C-terminus, which contains a putative PDZ binding site (Poliak etal, 1999), a feature shared by two other recently described members ofthe mammalian Caspr family, Caspr3 and Caspr4 (Spiegel et al, 2002). TheC-terminus of Caspr is rather unique compared to other members of thefamily in terms of its length, and the antibody the inventor has raisedis unlikely to cross react with the other Caspr isoforms. The C-terminusof Caspr does not have a putative PDZ binding motif, but shares a band4.1 binding domain with Caspr2 (Scherer and Arroyo, 2002). It would beinteresting to determine if this domain of Caspr mediates itsinteraction with Kv1.1 and Kv1.2.

Nogo-A Interactions in the CNS

The only known interacting partners of Nogo-A other than NgR are theNogo-interacting mitochondrial protein (NIMP) (Hu et al, 2002),α-tubulin, and MBP (Taketomi et al, 2002). All these interactions arelikely to involve the intracellular domains of Nogo-A, and not theNogo-66 extracellular loop, which is the Nogo-A domain most likely tofunction as an intercellular signaling ligand. The inventor's resultsindicate that the oligodendrocyte surface Nogo-66 loop binds directly toaxonal surface Caspr, thus implying a previously unsuspected function ofNogo-A at the axoglial junction. This interaction does not appear toinvolve NgR. In fact, it is unclear if any permanently recurringinteraction between adult oligodendroglial Nogo-A and axonal NgR isrequired under normal physiological conditions. The inventors believethat the Nogo-A/Caspr interaction may in some manner shape and maintainthe architecture of the axoglial junctions during and after myelination(FIG. 8). More specifically, this interaction may have a role inorganizing the location of other molecules at specific junction domains.

In summary, the inventor has shown that oligodendrocyte Nogo-A isclustered at specific axoglial junctions, where it interacts directlyvia its extracellular Nogo-66 loop with axonal Caspr, and indirectlywith K⁺ channel proteins. This represents the first NgR-independentNogo-66 interaction described to date, and has significant implicationsfor the role of Nogo-A in formation and maintenance of axoglial junctionarchitecture.

Materials and Methods Relating to the First Aspect of the Invention

Antibodies

The polyclonal antibody against Nogo-A was previously described (Liu etal, 2002). Two polyclonal antibodies against NgR were used in theinventor's experiments. One raised in rabbit with a glutathioneS-transferase (GST) fusion protein to amino acids 277-430 of human NgRand another a gift from Dr. Stephen Strittmatter (Yale University Schoolof Medicine, New Haven; Wang et al, 2002). Antibodies against F3 andNB-3 were described previously (Ang et al, 2001).

To raise polyclonal antibodies against Caspr, a 230 bp fragment encodingthe cytoplasmic region (amino acids 1308-1377) of human Caspr (Einheberet al, 1997) was amplified from human brain cDNA using primers5′-AGTCGGATCCACAAAATC ATCGA/CTAT/CA/CAGGG-3′ (forward) and5′-ACTCGAATTCAGACCTGGACT CCTCCTCCAA/GGATCTGG-3′ (reverse) with an addedBamH1 or EcoR1 site, respectively. The amplified fragment was digestedwith BamH1 and EcoR1 and subcloned in-frame into pGEX-3C, and thesequence of the final construct was verified by DNA sequencing. Theplasmid was transformed into E. coli BL21, and upon induction aCaspr-GST fusion protein of the expected size was recovered frombacterial lysates using glutathione-agarose beads. Caspr-GST was elutedfrom the beads using reduced glutathione, concentrated bylyophilization, and used to immunize rabbits. The immune serum obtainedfrom the rabbits was confirmed for its ability to recognize chick andmouse Caspr through immunoblotting and immunoprecipitation experiments.

Mouse monoclonal antibodies against Kv1.1 α-subunit (K20/78) and Na⁺channel (K58/35) were purchased from Upstate Biotechnology and Sigma,respectively. Polyclonal antibodies against Kv1.1, Kv1.2 and Kv2.1 werepurchased from Chemicon. The monoclonal anti-MAP2 was purchased fromSigma. The Cy2-conjugated goat anti-rabbit and Cy3-conjugated goatanti-mouse secondary antibodies were purchased from Amersham PharmaciaBiotech, and the ABC kit was purchased from Vector Laboratories.

EAE Model

The Experimental Autoimmune Encephalomyelitis (EAE) model in rats wasdeveloped according to a previous report (Ahn et al, 2001). In brief,Lewis rats (2 months old, female) received an injection of 0.5 ml/rat offresh rat spinal cord homogenate (SCH) in complete Freund's Adjuvant(CFA, containing 1 mg/ml Mycobacteria Tuberculosis; Sigma) (1:1) in thehind footpads bilaterally. Animals were closely observed for symptomsassociated with EAE to determine disease progression. At 13 dayspost-injection (dpi) ˜14 dpi, animals at the peak stage of EAE weresacrificed for further experiments.

Immunohistochemistry and Immunoelectron Microscopy

Wistar rats at different postnatal ages (P1, P5, P7, P14, P30, P60 andadults), CGT^(−/−) (P16 and P21; Coetzee et al, 1996) and Shiverer(adults; Jackson Laboratories) mice were perfused with 4%paraformaldehyde. The spinal cords and brainstems were removed andpost-fixed in 4% paraformaldehyde for 2 hr, and sequentially incubatedin 15% and 30% sucrose. Cryostat sections (10 μm) were double-labeledwith polyclonal antibodies to Nogo-A (1:200) or Caspr (1:200) inconjunction with a monoclonal Kv1.1 α-subunit (1:200) or Na⁺ channel(1:100) antibodies respectively, or with polyclonal NgR antibodies inconjunction with a monoclonal MAP2 (1:100) or Kv1.1 α-subunitantibodies. Immuno-stainings were visualized and photographed using aCarl Zeiss LSM5 confocal microscope. The lengths for Nogo-A and Kv1.1immuno-labeling and their overlaps at different postnatal days weremeasured. Co-localization for Nogo-A and Kv1.1, or Caspr/Kv1.1 atparanodes at different postnatal days was counted. In brainstem sectionsof normal and EAE rats, wild-type and shiverer mice, distances betweenpaired Kv1.1 immuno-labelings were measured and the Nogo-A clusters werecounted. Values were presented as mean±SEM. Statistical analyses werecarried out using the paired group T-test.

For electron microscopy, samples from adult Wistar rats and CGT^(−/−)(P16) were prepared according to published protocols (Huber et al,2002). Ultrathin sections of 90 nm thickness on nikel grids were blockedfor 40 min at room temperature with 1% BSA, 0.1% Tween 20, 1% normalgoat serum and 0.025% NaN₃ in 0.1 M sodium phosphate buffer (pH8.3),followed by overnight incubation at 4° C. with Nogo-A polyclonalantibodies in the same buffer, respectively. After washing thoroughlywith the above buffer, grids were incubated for 1 h with goatanti-rabbit IgG conjugated to 10 nm gold (1:20, Aurion) and fixed with2.5% aqueous glutaraldehyde for 15 min. After double staining withuranyl acetate and lead citrate, the grids were examined under a Philips208 electron microscope.

Western Blot and Co-Immunoprecipitation Assays

From adult, postnatal day 1 (P1) to 30 (P30) Wistar rats, variousregions of the CNS (total brain, brainstem, hippocampus, cerebral cortexand spinal cord), spinal cords from EAE and control rats, were harvestedand extracted in phosphate-buffered saline containing 1% Triton X-100and a cocktail of protease inhibitors. Lysates were electrophoresed onSDS-PAGE gel and blotted onto nitrocellulose membranes (Hybond C-extra,Amersham). Identical blots were probed with antibodies against Nogo-A,Caspr, Nogo-66 Receptor (NgR) and γ-tubulin (for loading normalization),and visualized with the Pierce chemiluminescent detection reagents.

For co-immunoprecipitation (IP) experiments, brain membrane fractionswere prepared as described previously (Lei et al, 2002). Briefly, braintissue was homogenized in ice-cold homogenizing buffer (320 mM sucrose,10 mM Tris-HCl pH7.4, 1 mM NaHCO₃ pH7.4, 1 mM MgCl₂) supplemented with1% protease inhibitor cocktail (Amersham), and subsequently centrifugedat 5,000 g for 15 min. The supernatant was collected and spun at 60,000g (Beckman ultracentrifuge) for 60 min at 4° C. Pellets were thendissolved in a lysis buffer (10 mM Tris-HCl pH9, 150 mM NaCl, 0.5%Triton X-100, 1% sodium deoxycholate (DOC), 0.5% SDS, 2 mM EDTA, and 1%protease inhibitor cocktail) for subsequent experiments.Immunoprecipitated proteins, using of non-immune IgG, Caspr, Nogo-A andNB3 antibodies, respectively, were eluted from the beads with Laemmlisample buffer and separated on 8% SDS-PAGE gels, prior to beingtransferred to nitrocellulose membrane and probed for Caspr, Nogo-A,Kv1.1, Kv1.2, F3, or NB3. In separate experiments, a Nogo-A expressionconstruct in the mammalian expression vector pCIneo (Promega) wastransiently transfected into Caspr/F3-expressing CHO (Faivre-Sarraih etal, 2000), F3-expressing CHO (Gennarini et al, 1991) or wild type CHOcells for co-IP studies.

Cell Adhesion Assay and PI-PLC Treatment

The Nogo-66 peptide (KLSDVLDDVLFLRRLEKITCNVHGLASNSYKQVLEESIAVESELYARFPHGEDSKQIAQIVGKYIR) was purchased from Loke Diagnostics ApS(Denmark). To generate recombinant proteins of Nogo-66-GST andNogo-N-terminal-GST (Nogo-N-GST), the encoding sequences for Nogo-66 andNogo-N-terminus were amplified from human brain cDNA clone HK07722(Nogo-A) using the primer sets below: 5′-CTGAATTCTTAGGATATACAAGGGTGT-3′(forward) 5′-GCTAAGCTTTCACTTCAGAGAATCAACTA-3′ (reverse) for Nogo- 66-GST5′-AGGAATTCTAGATGAGACCCTTTTTGC-3′ (forward)5′-CCCAAGCTTTCAATTAAAACTGTCTTTTGCTTT-3′ (reverse) for Nogo- N-GST.

The PCR products were digested with EcoRI and HindIII and ligated intoEcoRI/Hind III-digested pGEX-KG (Guan and Dixon, 1991). Then, theserecombinant plasmids were transformed into E. coli Top 10 cells. GSTfusion proteins were recovered from the bacterial lysates and purifiedusing glutathione-agarose beads. The cell adhesion assay was carried outas previously described (Xiao et al, 1996). Protein spots (1.5 μl of 5μM GST, Nogo-66-GST or 100 μM Nogo-66) were applied ontonitrocellulose-coated surfaces of the Petri dishes (Becton Dickinson)and incubated for 2 hours at 37° C. in a humidified atmosphere. Thedishes were then incubated overnight with PBS containing 2%heat-inactivated fatty acid-free BSA (Sigma) to block residualnon-specific protein binding sites. Mock-transfected CHO, F3-transfectedor Caspr/F3 co-transfected CHO cells were then plated in 2 ml ofchemically defined medium at a density of 2.5×10⁵ cells/ml and incubatedat 37° C. in a humidified atmosphere. After 12 hours, cells were fixedby flooding with PBS containing 2.5% glutaraldehyde. Cells adhering tothe various spots were photographed and counted. All experiments wereperformed at least three times. Statistical analysis was carried out byStudent-t test. The level of significance was chosen at p<0.05.

Where indicated, Caspr/F3-transfected CHO cells were treated with PI-PLC(0.02, 0.04 or 0.06 U/ml) (Sigma), incubated for 2 hours and then platedinto the dishes following the same procedures as described above. PI-PLCtreated cells were subjected to Western blot analysis andimmunochemistry for F3/Contactin as described above.

GST Pull-Down Assay

The bead-bound GST, GST-Nogo-66 and GST-Nogo-N (20 μg) were mixedrespectively with 1.5 ml of membrane extracts of adult brain or of wildtype, F3-expressing and Caspr/F3-expressing CHO cells that had beentransiently transfected with Kv1.1 (a gift from Dr. Trimmer, Nakahira Ket al, 1996) in 0.1 M sodium phosphate buffer (pH7.4) and incubatedovernight at 4° C. The bound proteins were eluted with 2× Laemmli samplebuffer, separated by SDS-PAGE and probed with Caspr and Kv1.1antibodies.

Lipid Rafts Analysis

Preparation of detergent extracts from rat cerebral cortex (15 days andadult) was carried out primarily according to the procedures describedby Kramer et al, 1999. In brief, dissected rat cerebral cortex (2 g wetweight total) was homogenized in 12.5 ml TBS (pH 7.4) containing 2%Triton-X 100, 2 mM pervanadate, protease inhibitor tablet (Roche) andstirred for 30 min at 4° C. The detergent extracts were adjusted to 40%sucrose by adding equal volumes of 80% sucrose in TBS and placed in theultracentrifuge tube for the SW28 rotor. A linear gradient from 5% to30% sucrose in TBS were layered onto the lysate sample. Gradients werecentrifuged for 18 hrs at 25,000 rpm. Two ml fractions were collectedfrom top to bottom. Proteins in each fraction were analyzed by SDS-PAGEfollowed by Western blot. Detergent insoluble floating materials weremostly recovered in fraction 5.

SECOND ASPECT OF THE INVENTION Background Information to the SecondAspect of the Invention

Myelination is a complex multistep process where the underlyingmolecular mechanism remains far from being completely defined. However,it is believed that the interaction between axons and myelin competentcells at the paranode play an important role in the insulation of axonalsegments in spiral wraps of myelin. This axo-glial contact has beenlikened to invertebrate septate junctions and has been proposed to serveas an anchor point between axons and myelin loops, to act as a partialdiffusion barrier into the periaxonal space and to segregate the axoninto domains by preventing the lateral diffusion of membrane components(Rosenbluth, 1995). In recent years, specific molecules have been foundto be located at the paranodal region.

F3/contactin is a glycosyl-phosphatidylinositol (GPI) linked molecule ofthe immunoglobulin superfamily of neural cell adhesion molecules(Gennarini et al, 1989; Faivre-Sarrailh et al, 2000). The molecule iscomposed of a string of modular immunoglobulin domains and fibronectintype III repeats. The inventor previously (Xiao et al, 1998 incorporatedherein by reference) demonstrated that F3 is a neuronal receptor for theextracellular matrix glycoprotein tenascin-R, a glia-derived moleculespecifically located at nodes of Ranvier (Wintergerst et al, 1993). Thebinding site on tenascin-R, in its interaction with F3, was localized tothe EGF (epidermal growth factor) like repeats (Xiao et al, 1996, 1997,incorporated herein by reference). Tenascin-R is a functional modulatorof sodium channel subunits. F3 interacts in trans with RPTPζ/p (receptorprotein tyrosine phosphatase) to promote neurite outgrowth (Sakurai etal, 1997) and in cis with RPTPα (Zeng et al, 1999) to transduceextracellular signals to myelination-related Fyn kinase (Umemori et al,1994). Additionally, F3 co-localizes and interacts in cis withCaspr/Paranodin and in trans with glial neurofascin 155 at the paranode(Girault and Peles, 2002), a key site of axoglial contact formyelination. F3 null mice exhibit partially disrupted paranodalstructure and die by P18 (Berglund et al, 1999), suggesting that F3 maybe critical for development.

F3-associated protein (Caspr), also known as paranodin, is an additionalaxonal component of the paranode (Einheber et al, 1997; Menegoz et al,1997). Rios et al (2000) further showed that F3 and Caspr co-localizeand interact in a cis fashion at the paranode during myelination. Inadult sciatic and optic nerves, F3 staining localized to the paranodes,although staining also extended to the nodes in the optic nerve.

NB-3 is a neural cell adhesion molecule in the same subfamily as F3 (Leeet al, 2000). In the cerebrum, NB-3 mRNA analysis revealed a low levelof expression during embryogenesis with an abrupt increase in thepostnatal period, reaching a maximum level in the postnatal seventh daywhich corresponds to the time frame for myelination. Subsequently,levels decreased to one-fifth of the peak and remained so in adulthood.

Myelination in the vertebrate central nervous system (CNS) is essentialfor rapid impulse conduction. In the CNS, oligodendrocyte (OL)differentiation is mediated by neuron derived signals (Barres and Raff,1999). Jagged1/Notch1, an axoglial interaction, promotesoligodendrocytes precursor cell (OPC) migration and inhibits OPCdifferentiation (Wang et al, 1998). Notch/Jagged1 signaling pathwayplays a critical role in promoting gliogenesis, such as radial glialcells in the fetal forebrain, Schwann cells in dorsal root ganglia, andMüller glial cells in the retina (Furukawa et al, 2000; Hojo et al,2000; Gaiano et al, 2000; Morrison et al, 2000; Wakamatsu et al, 2000;Tanigaki et al, 2001). Conditional ablation of Notch1 in OPCs results inthe appearance of ectopic premature OLs and subsequent apoptosis (Genoudet al, 2002) and the failure of efficient remyelination is partlyattributed to the activation of OPC Notch receptor byastrocyte-expressed Jagged1 in multiple sclerosis (John et al, 2002)indicating that OPC differentiation could be disordered when Notch1 isabsent or inadequately activated by Jagged1. Thus, other pathways viaNotch1, besides the inhibitory Jagged1/Notch1 signalling pathway, mayinstructively mediate OPC differentiation into OLs. However, themolecular mechanisms controlling the timing of OPC differentiation toOLs and the subsequent OL maturation remain poorly defined. Given thatJagged1 is downregulated significantly before the maturation ofoligodendrocytes, it is conceivable that other molecules may interactwith Notch, which continually plays a role in myelination. Moleculescongregated at distinct segments of the axon are potential Notch1ligands.

Notch is a type I transmembrane protein mediating cell fate selectionvia lateral inhibition. Its core signaling mechanism involves RegulatedIntramembrane Proteolysis (RIP) (Ebinu and Yankner, 2002). Upon bindingthe classic ligands, Delta, Serrate/Jagged and Lag-2 (collectivelycalled DSL), Notch undergoes two proteolytic cleavages that release itsintracellular domain (NICD). NICD translocates to the nucleus andinteracts with RBP-J transcription factor to activate, for instance, Hesgenes (Martinez Arias et al, 2002). In addition, Deltex¹ (DTX1) has beenidentified as a cytoplasmic downstream element of the Notch signalingpathway. DTX homologs share three common domains, namely, the N-terminalregion, proline-rich and RING-H2 finger motifs (Kishi et al, 2001).Particularly, the N-terminal region interacts with NICD. Notch signalingvia DTX1 represses JNK signaling, a pathway regulating OLdifferentiation, and cooperates with Wingless signaling (Brennan andGardner, 2002; Martinez Arias et al, 2002). Although several studiesimply the existence of an extracellular ligand that activates Notch/DTX1signaling, the putative ligand has not yet been identified.

Notch has been shown to regulate glial differentiation (Wang et al,1998; Gaiano et al, 2000; Morrison et al, 2000). Of significance to theinventor was that the Notch extracellular portion contains, as doestenascin-R, multiple EGF-like repeats which are sites of potentialligand-receptor interactions (Rebay et al, 1991). It thus becomesconceivable to the inventor that glia-derived Notch could be a bindingpartner of axonal F3 and NB-3. In the rat optic nerve, Wang et al (1998)demonstrate Jagged1 expression on retinal ganglion cell axons. Jagged1signals to Notch on oligodendrocyte precursors to inhibit theirdifferentiation. Of interest is the expression pattern of Jagged1 whichbecomes developmentally downregulated with a time course that parallelsmyelination (Dugas et al, 2001). This led to the conclusion that Jagged1signals to oligodendrocytes thus as part of a localized timing mechanismto regulate oligodendrocyte differentiation and thus myelination. Buthow is the segmental nature of the myelin sheath preserved? What is thestop signal that prevents myelinating oligodendrocytes from encroachingupon putative nodes of Ranvier? The inventor hypothesized that suchaxonal stop signals should logically exist on either side of the nodes,namely at the paranodes. As detailed below, the inventor has indeeddemonstrated that F3 is able to act as a stop signal for oligodendrocyteprocesses and that NB-3 may participate in the triggering ofoligodendrocyte differentiation.

Summary of the Second Aspect of the Invention

The present inventor has for the first time shown that both F3 and NB-3are physiological ligands of the oligodendroglial Notchreceptor—establishing the presence of a signalling pathway via Deltex1to co-ordinate events during myelination.

Thus, the inventor has identified a new paranodal molecule—NB-3 andshowed that stop signals are located at paranodes which involve F3/NB-3signalling to Notch on the surface of oligodendroglia. In a co-culturesystem between OLN-93 cells and F3-transfected CHO cells,oligodendroglial cellular processes terminate and spread over theF3-transfected cell bodies but bypass control CHO cells. Cell adhesion,co-immunoprecipitation and GST pull-down assays confirm that F3/NB-3 andNotch associate as a complex. MAG becomes upregulated when OLN-93 cellsand F3-transfected CHO cells are co-cultured and when OLN-93 cells andprimary oligodendrocytes contact F3 and NB-3 protein substrates. Theseresults describe a novel and functionally significant signallinginteraction between F3/NB-3 and Notch that is involved in the regulationof myelination. The present inventor also shows in cell adhesion testsand biochemical assays that F3 is able to bind to Notch1 and Notch2.Furthermore the interaction induces radical morphological change inoligodendrocyte cell line OLN-93 which develops the ensheathing featureand significantly upregulates myelin-related proteins, such as MAG(myelin-associated glycoprotein), CNPase (2′,3′-cyclic nucleotide3′-phosphodiesterase), and PLP. These results suggest that F3 is aphysiological ligand of Notch receptor and the signaling plays animportant role in oligodendrocyte maturation.

This determination shows that F3 and NB-3 interaction with Notch playsan important role in oligodendrocyte maturation.

The inventor has further determined that F3 induces Notch intramembranecleavage at the S3 site and that F3/Notch-induced MAG upregulation isindependent of HES1 and involves Deltex 1 (DTX1). DTX1 is a knowncytoplasmic downstream element of the Notch signaling pathway. It haspreviously been shown that Notch signaling via DTX1 represses JNKsignalling, a pathway that regulates oligodendrocyte differentiation.However, the previous studies have not identified the extracellularligand that activates Notch/DTX1 signaling.

Here the inventor reports that NB-3 is a neuron-derived cell recognitionmolecule developmentally clustering at the CNS paranodes and identifyNB-3 as a functional ligand of Notch1. The NB-3/Notch signalling pathwaypromotes OPC differentiation and OL maturation via Deltex1. Moreover,Jagged1 is localized to juxtaparanodes and internodes. Thus a spatialsignal switch mechanism from Jagged1/Notch1 to NB-3/Notch1 may existalong the axon, which coordinates oligodendroglial maturation.

Neurons and glia in the vertebrate CNS arise in temporally distinct,albeit overlapping phases. Neurons are generated first, followed byastrocytes and then oligodendrocytes (OLs) from common progenitor cells(Sauvageot and Stile, 2002). Increasing evidence indicates thataxon-derived signals are temporally and spatially required formodulating OL maturation as well as myelin formation (Barres and Raff,1999; Hu et al, 2003). However, little is known about how neuronalmolecules participate in OL generation from neural stem cells (NSCs).The inventor's investigation on how to favorably direct embryonic neuralstem cells to differentiate into OLs showed that NB-3, a neuronal cellrecognition molecule, bound to Notch1 and triggered nucleartranslocation of the Notch1 intracellular domain in the stem cells. ThisNB-3/Notch1 interaction promotes oligodendrogliogenesis from embryonicneural stem cells, which can be blocked by dominant-negative Notch1 anddeletion mutants of Deltex1 that lacks the Ring-H2 motif. However,constitutively active Notch1 alone fails to promote OL generation,suggesting that NB-3-induced NICD is required in this event. Takentogether, the observations here demonstrate that the NB-3/Notch1signalling pathway via Deltex1 instructs oligodendrogliogenesis.

Thus the present invention provides a method of stimulatingdifferentiation of an oligodendrocyte or precursor thereof, comprisingcontacting said oligodendrocyte or precursor with F3, NB-3, or a mimeticof either.

The present invention further provides a method of stimulatingmyelination of a neuron, specifically a neural axon, comprisingcontacting an oligodendrocyte, a precursor thereof, or a neuron, withF3, NB-3, or a mimetic of either.

In either of the methods described above, the oligodendrocyte,precursor, or neuron as appropriate, is preferably contacted with bothF3 and NB-3, or mimetics thereof. F3 and NB-3 may be present as acomplex.

Expression of myelin proteins, such as MAG, will typically beupregulated in said oligodendrocyte or precursor in response to thecontacting step.

Without wishing to be bound by any particular theory, it is believedthat upregulation of MAG is induced through binding of F3, NB-3 ormimetics thereof to Notch, particularly to Notch 1 or 2, on the surfaceof the oligodendrocyte or precursor thereof. Such binding is believed toinduce Notch signalling, via Deltex-1.

The oligodendrocyte precursor may be an oligodendroglial precursor cell(OPC) or a neural stem cell (NSC). OPCs typically display CNpase, Gal Cand MAG on their surface; NSCs display Nestin marker. OPCs and NSCs aredescribed by Wang et al, 1998 (Notch receptor activation inhibitsoligodendrocyte differentiation. Neuron 21, 63-75); Morrison S. J., 2001(Neuronal potential and lineage determination by neural stem cells.Curr. Opin. Cell Biol. 13, 666-672); Sauvageot, C. M. and Stiles, C. D.,2002 (Molecular mechanism controlling cortical gliogenesis. Curr. Opin.Neurobiol. 12, 244-249); Xiao et al, 2003 (F3/Contactin is a notchligand. Cell, Vol 115, 163-175).

The methods may be performed in vitro or ex vivo. The methods areparticularly applicable to the generation of differentiatedoligodendrocytes which may be used for therapeutic purposes. Thus, aftersaid contacting step, the OPC or NSC may be introduced or implanted intoa subject having disease of, or injury to, the central nervous system;for example, any condition characterised by demyelination of neurons,such as multiple sclerosis (MS), epilepsy, stroke and spinal cord injury(SCI). Following introduction or implantation, these treated cells maycontinue to differentiate and provide myelination for de-myelinatedneurons.

It may be desirable to obtain the oligodendrocyte precursor cells fromthe subject to whom they are to be administered after treatment.

The invention further provides a composition comprising F3 and NB-3, ormimetics thereof, in combination with a carrier. The composition maycomprise a complex between F3 and NB-3, or a mimetic thereof.

In certain embodiments, the composition is a pharmaceutical composition,and so comprises a pharmaceutically accceptable carrier. Preferablypharmaceutical compositions are formulated for injection in vivo, andstill more preferably for direct injection into the CNS.

Also provided are compositions as described above for use in a method ofmedical treatment. In particular the compositions are provided for usein the treatment of injury to, or disease of, the CNS. They may be usedfor treatment of any condition characterised by demyelination ofneurons, such as SCI, MS, epilepsy or stroke.

Also provided is the use of F3 and/or NB-3 in the preparation of amedicament for the treatment of injury or disease to the CNS. They maybe formulated individually or separately. Separately formulated NB-3 andF3 may nevertheless be administered together.

Thus there is also provided the use of F3 in the preparation of amedicament for the treatment of injury or disease to the CNS, whereinthe medicament is for administration in combination with NB-3 or amimetic thereof.

Likewise, there is provided the use of NB-3 in the preparation of amedicament for the treatment of injury or disease to the CNS, whereinthe medicament is for administration in combination with F3 or a mimeticthereof.

The invention further provides a method of stimulating myelination of aneuron, specifically a neural axon, comprising contacting a neuron or anoligodendroglial cell with a composition as described herein.

The invention further provides a method of treating a subject havingdisease of, or injury to, the central nervous system, comprisingadministering to the subject a pharmaceutical composition as describedherein.

As will be clear from the foregoing, the subject will typically have acondition characterised by demyelination, such as SCI, MS, epilepsy orstroke.

The invention further provides a method of screening for a substancecapable of modulating (preferably promoting) interaction between Notchand F3 and/or NB-3, the method comprising contacting F3 and/or NB-3,Notch and a candidate substance, and determining the interaction betweenNotch and F3 and/or NB-3.

The method may further comprise contacting Notch and F3 and/or NB-3 inthe absence of the candidate substance under otherwise analogousconditions, and determining the interaction between Notch and F3 and/orNB-3.

Preferably the method comprises contacting a complex between Notch andF3 and/or NB-3 with the candidate substance. The complex is preferablyformed before it is contacted with the candidate substance.

The method may be performed by any appropriate method. The skilledperson will be well aware of many suitable assay formats, and will becapable of designing further examples.

Any or all of NB-3, F3 and Notch may be present in, or on, a cell. Thegene from which the protein is expressed may be endogenous to the cellin question, or it may be present on a vector introduced into the cell.The protein is preferably expressed on the surface of the cell.

Additionally or alternatively, any or all of NB-3, F3 and Notch may beimmobilised on a solid support. One or both may comprise a detectablelabel as described in more detail below.

In particular embodiments, Notch is present on a cell surface, and themethod comprises determining Notch signalling, particularly viaDeltex 1. If the cell in question is an oligodendrocyte or precursorthereof, the method may comprise determination of upregulation of MAGexpression.

The invention further provides a method of manufacturing apharmaceutical formulation comprising, having identified a substancecapable of modulating interaction between Notch and F3 and/or NB-3 by ascreening method described herein, the further step of formulating saidsubstance with a pharmaceutically acceptable carrier. The method maycomprise the further step of optimising said identified substance foradministration in vivo prior to formulation.

In specific embodiments, the present invention firstly provides a methodfor enhancing myelination in an individual comprising administering tosaid patient an activating agent of Notch receptor, said activatingagent comprising F3, NB-3, or a mimetic thereof.

The method is preferably used in the treatment of SCI, MS, epilepsy orstroke.

The invention also provides a pharmaceutical composition comprising anactivating agent of a Notch receptor, said activating agent comprisingF3, NB-3, or a mimetic thereof.

Also provided is a method of screening for substances capable ofmodulating the interaction between a ligand and a Notch receptor, theligand being F3, NB-3, or a mimetic thereof, comprising contacting asubstance with the ligand and the receptor; determining the interactionbetween the ligand and the receptor and comparing this with theinteraction between the receptor and ligand under comparable conditionsbut in the absence of said substance.

The method may further comprise producing a pharmaceutical compositioncontaining said substance.

Cells

The term oligodendrocyte is used herein to refer to oligodendroglialcells capable of laying down a myelin sheath around a neuronal axon inthe central nervous system (CNS).

The term oligodendrocyte precursor cell is used to refer to cellscapable of differentiating into oligodendrocytes on administration ofsuitable stimuli, such as F3 and NB-3. Such cells includeoligodendroglial precursor cells (OPC) and neural stem cells (NSC).

Protein Sequences

The term “Notch” is used to encompass all isoforms of the Notch protein,including Notch 1, 2 and 3, as well as portions and isolated domainsthereof, such as the extracellular domain, as well as mutants andvariants thereof having greater than 80%, 85%, 90%, 95%, 96%, 97% 98% or99% identity to the sequence given below. Preferred proteins are Notch 1and 2. Orthologous proteins from other mammalian species are alsoincluded. Preferably the Notch protein has the ability to bind to a NB-3and/or a F3 protein. It may also have the ability to induce upregulationof MAG protein expression via Deltex 1 signalling in an oligodendrocyteor precursor thereof.

The term “F3” is used to encompass isoforms of the F3 protein, andisolated domains of such proteins, as well as mutants and variantsthereof having greater than 80%, 85%, 90%, 95%, 96%, 97% 98% or 99%identity to the sequence given below. Orthologous proteins from othermammalian species are also included. The F3 protein preferably has theability to bind to the extracellular domain of a Notch protein,particularly of Notch 1 and 2.

The term “NB-3” is used to encompass isoforms of the NB-3 protein, andisolated domains of such proteins, as well as mutants and variantsthereof having greater than 80%, 85%, 90%, 95%, 96%, 97% 98% or 99%identity to the sequence given below. Orthologous proteins from othermammalian species are also included. The NB-3 protein preferably has theability to bind to the extracellular domain of a Notch protein,particularly of Notch 1 and 2.

The amino acid sequences of Notch, F3 and NB-3 proteins are shown below,along with their GenBank accession numbers; F3/Contactin gi:414791(CAA79696) 1 mkmwllvshl viisittcla eftwyrrygh gvseedkgfg pifeeqpintiypeeslegk 61 vslncraras pfpvykwrmn ngdvdltsdr ysmvggnlvi nnpdkqkdagiyyclasnny 121 gmvrsteatl sfgyldpfpp eerpevrvke gkgmvllcdp pyhfpddlsyrwllnefpvf 181 itmdkrrfvs qtngnlyian veasdkgnys cfvsspsitk svfskfiplipiperttkpy 241 padivvqfkd vyalmgqnvt lecfalgnpv pdirwrkvle pmpstaeistsgavlkifni 301 qledegiyec eaenirgkdk hqariyvqaf pewvehindt evdigsdlywpcvatgkpip 361 tirwlkngya yhkgelrlyd vtfenagmyq ciaentygai yanaelkilalaptfemnpm 421 kkkilaakgg rviieckpka apkpkfswsk gtewlvnssr iliwedgsleinnitrndgg 481 iytcfaennr gkanstgtlv itdptriila pinaditvge natmqcaasfdpaldltfvw 541 sfngyvidfn kenihyqrnf mldsngelli rnaqlkhagr ytctaqtivdnssasadlvv 601 rgppgppggl riediratsv altwsrgsdn hspiskytiq tktilsddwkdaktdppiie 661 gnmeaaravd lipwmeyefr vvatntlgrg epsipsnrik tdgaapnvapsdvgggggrn 721 reltitwapl sreyhygnnf gyivafkpfd geewkkvtvt npdtgryvhkdetmspstaf 781 qvkvkafnnk gdgpysllav insaqdapse aptevgvkvl ssseisvhwehvlekivesy 841 qirywaahdk eeaanrvqvt sqeysarlen llpdtqyfie vgacnsagcgppsdmieaft 901 kkappsqppr iissvrsgsr yiitwdhvva lsnestvtgy kvlyrpdgqhdgklysthkh 961 sievpiprdg eyvvevrahs dggdgvvsqv kisgaptlsp sllglllpafgilvylef NB3 gi:5631291 (BAA82612) 1 mrllwklvil lplinssagd gllsrpiftqephdvifpld lsksevilnc aangypsphy 61 rwkqngtdid ftmsyhyrld ggslainsphtdqdigmyqc latnllgtil srkaklqfay 121 iedfetktrs tvsvregqgv vllcgppphfgdlsyawtfn dnplyvqedn rrfvsqetgn 181 lyiakvepsd vgnytcfitn keaqrsvqgpptplvqrtdg vmgeyepkie vrfpetiqaa 241 kdssvklecf algnpvpdis wrrldgsplpgkvkysksqa ileipnfqqe degfyecias 301 nlrgrnlakg qlifyappew eqkiqnthlsiydnllweck asgkpnpwyt wlkngerlnp 361 eeriqiengt liitmlnvsd sgvyqcaaenkyqiiyanae lrvlasapdf skspvkkksf 421 vqvggdivig ckpnafpraa iswkrgtetlrqskriflle dgslkiynit rsdagsytci 481 atnqfgtakn tgslivkert vitvppskmdvtvgesivlp cqvshdpsie vvfvwffngd 541 vidlkkgvah feriggesvg dlmirniqlhhsgkylctvq ttleslsava diivrgppgp 601 pedvqvedis sttsqlswra gpdnnspiqiftiqtrtpfs vgwqavatvp eilngktyna 661 tvvglspwve yefrvvagns igigepsepsellrtkasvp vvapvnihgg ggsrselvit 721 wesipeelqn gegfgyiimf rpvgsttwskekvssvessr fvyrnesiip lspfevkvgv 781 ynnegegsls tvtivysged epqlaprgtslqsfsaseme vswnaiawnr ntgrvlgyev 841 lywtddskes migkirvsgn vttknitglkantiyfasvr ayntagtgps sppvnvttkk 901 sppsqppani awkltnsklc lnwehvktmenesevlgyki lyrqnrqskt hiletnntsa 961 ellvpfeedy lieirtvsdg gdgssseeiripkmsslssr giqflepsth flsivivifh 1021 cfaiqpli Notch1 gi:11275980(AAG33848) 1 mppllapllc lallpalaar gprcsqpget clnggkceaa ngteacvcggafvgprcqdp 61 npclstpckn agtchvvdrr gvadyacsca lgfsgplclt pldnacltnpcrnggtcdll 121 tlteykcrcp pgwsgkscqq adpcasnpca nggqclpfea syichcppsfhgptcrqdvn 181 ecgqkprlcr hggtchnevg syrcvcrath tgpncerpyv pcspspcqnggtcrptgdvt 241 hecaclpgft gqnceenidd cpgnnckngg acvdgvntyn cpcppewtgqyctedvdecq 301 lmpnacqngg tchnthggyn cvcvngwtge dcseniddca saacfhgatchdrvasfyce 361 cphgrtgllc hlndacisnp cnegsncdtn pvngkaictc psgytgpacsqdvdecslga 421 npcehagkci ntlgsfecqc lqgytgprce idvnecvsnp cqndatcldqigefqcmcmp 481 gyegvhcevn tdecasspcl hngrcldkin efqcecptgf tghlcqydvdecastpckng 541 akcldgpnty tcvctegytg thcevdidec dpdpchygsc kdgvatftclcrpgytghhc 601 etninecssq pcrlrgtcqd pdnaylcfcl kgttgpncei nlddcasspcdsgtcldkid 661 gyecacepgy tgsmcnsnid ecagnpchng gtcedgingf tcrcpegyhdptclsevnec 721 nsnpcvhgac rdslngykcd cdpgwsgtnc dinnnecesn pcvnggtckdmtsgivctcr 781 egfsgpncqt ninecasnpc lnkgtciddv agykcncllp ytgatcevvlapcapspcrn 841 ggecrqsedy esfscvcpta gakgqtcevd inecvlspcr hgascqnthgxyrchcqagy 901 sgrncetdid dcrpnpchng gsctdginta fcdclpgfrg tfceedinecasdpcrngan 961 ctdcvdsytc tcpagfsgih cenntpdcte sscfnggtcv dginsftclcppgftgsycq 1021 hvvnecdsrp cllggtcqdg rglhrctcpq gytgpncqnl vhwcdsspcknggkcwqtht 1081 qyrcecpsgw tglycdvpsv scevaaqrqg vdvarlcqhg glcvdagnthhcrcqagytg 1141 sycedlvdec spspcqngat ctdylggysc kcvagyhgvn cseeideclshpcqnggtcl 1201 dlpntykcsc prgtqgvhce invddcnppv dpvsrspkcf nngtcvdqvggysctcppgf 1261 vgercegdvn eclsnpcdar gtqncvqrvn dfhcecragh tgrrcesvingckgkpckng 1321 gtcavasnta rgfickcpag fegatcenda rtcgslrcln ggtcisgprsptclclgpft 1381 gpecqfpass pclggnpcyn qgtceptses pfyrclcpak fngllchildysfgggagrd 1441 ippplieeac elpecqedag nkvcslqcnn hacgwdggdc slnfndpwknctqslqcwky 1501 fsdghcdsqc nsagclfdgf dcqraegqcn plydqyckdh fsdghcdqgcnsaecewdgl 1561 dcaehvperl aagtlvvvvl mppeqlrnss fhflrelsrv lhtnvvfkrdahgqqmifpy 1621 ygreeelrkh pikraaegwa apdallgqvk asllpggseg grrrreldpmdvrgsivyle 1681 idnrqcvqas sqcfqsatdv aaflgalasl gslnipykie avqsetveppppaqlhfmyv 1741 aaaafvllff vgcgvllsrk rrrqhgqlwf pegfkvseas kkkrreplgedsvglkplkn 1801 asdgalmddn qnewgdedle tkkfrfeepv vlpdlddqtd hrqwtqqhldaadlrmsama 1861 ptppqgevda dcmdvnvrgp dgftplmias csgggletgn seeeedapavisdfiyqgas 1921 lhnqtdrtge talhlaarys rsdaakrlle asadaniqdn mgrtplhaavsadaqgvfqi 1981 lirnratdld armhdgttpl ilaarlaveg mledlinsha dvnavddlgksalhwaaavn 2041 nvdaavvllk ngankdmqnn reetplflaa regsyetakv lldhfanrditdhmdrlprd 2101 iaqermhhdi vrlldeynlv rspqlhgapl ggtptlsppl cspngylgslkpgvqgkkvr 2161 kpsskglacg skeakdlkar rkksqdgkgc lldssgmlsp vdslesphgylsdvasppll 2221 pspfqqspsv plnhlpgmpd thlgighlnv aakpemaalg gggrlafetgpprlshlpva 2281 sgtstvlgss sggalnftvg gstslngqce wlsrlqsgmv pnqynplrgsvapgplstqa 2341 pslqhgmvgp lhsslaasal sqmmsyqglp strlatqphl vqtqqvqpqnlqmqqqnlqp 2401 aniqqqqslq ppppppqphl gvssaasghl grsflsgeps qadvqplgpsslavhtilpq 2461 espalptslp sslvppvtaa qfltppsqhs ysspvdntps hqlqvpehpfltpspespdq 2521 wssssphsnv sdwsegvssp ptsmqsqiar ipeafk Notch2gi:11275978 (AAA36377) 1 mpalrpallw allalwlcca apahalqcrd gyepcvnegmcvtyhngtgy ckcpegflge 61 ycqhrdpcek nrcqnggtcv aqamlgkatc rcasgftgedcqystshpcf vsrpclnggt 121 chmlsrdtye ctcqvgftgk ecqwtdacls hpcangstcttvanqfsckc ltgftgqkce 181 tdvnecdipg hcqhggtcln lpgsyqcqcp qgftgqycdslyvpcapspc vnggtcrqtg 241 dftfecnclp gfegstcern iddcpnhrcq nggvcvdgvntyncrcppqw tgqfctedvd 301 ecllqpnacq nggtcanrng gygcvcvngw sgddcseniddcafasctpg stcidrvasf 361 scmcpegkag llchlddaci snpchkgalc dtnplngqyictcpqgykga dctedvdeca 421 mansnpceha gkcvntdgaf hceclkgyag prcemdinechsdpcqndat cldkiggftc 481 lcmpgfkgvh celeinecqs npcvnngqcv dkvnrfqclcppgftgpvcq ididdcsstp 541 clngakcidh pngyecqcat gftgvlceen idncdpdpchhgqcqdgids ytcicnpgym 601 gaicsdqide cysspclndg rcidlvngyq cncqpgtsgvnceinfddca snpcihgicm 661 dginryscvc spgftgqrcn ididecasnp crkgatcingvngfrcicpe gphhpscysq 721 vneclsnpci hgnctgglsg ykclcdagwv gincevdkneclsnpcqngg tcdnlvngyr 781 ctckkgfkgy ncqvnideca snpclnqgtc fddisgytchcvlpytgknc qtvlapcspn 841 pcenaavcke spnfesytcl capgwqgqrc tidideciskpcmnhglchn tqgsymcecp 901 pgfsgmdcee diddclanpc qnggscmdgv ntfsclclpgftgdkcqtdm neclsepckn 961 ggtcsdyvns ytckcqagfd gvhcennine ctesscfnggtcvdginsfs clcpvgftgs 1021 fclheinecs shpclnegtc vdglgtyrcs cplgytgkncqtlvnlcsrs pcknkgtcvq 1081 kkaesqclcp sgwagaycdv pnvscdiaas rrgvlvehlcqhsgvcinag nthycqcplg 1141 ytgsyceeql decasnpcqh gatcsdfigg yrcecvpgyqgvnceyevde cqnqpcqngg 1201 tcidlvnhfk cscppgtrgl lceeniddca rgphclnggqcmdriggysc rclpgfager 1261 cegdinecls npcssegsld ciqltndylc vcrsaftgrhcetfvdvcpq mpclnggtca 1321 vasnmpdgfi crcppgfsga rcqsscgqvk crkgeqcvhtasgprcfcps prdcesgcas 1381 spcqhggsch pqrqppyysc qcappfsgsr celytappstppatclsqyc adkardgvcd 1441 eacnshacqw dggdcsltme npwancsspl pcwdyinnqcdelcntvecl fdnfecqgns 1501 ktckydkyca dhfkdnhcnq gcnseecgwd gldcaadqpenlaegtlviv vlmppeqllq 1561 darsflralg tllhtnlrik rdsqgelmvy pyygeksaamkkqrmtrrsl pgeqeqevag 1621 skvfleidnr qcvqdsdhcf kntdaaaall ashaiqgtlsyplvsvvses ltpertqlly 1681 llavavviil fiillgvima krkrkhgslw lpegftlrrdasnhkrrepv gqdavglknl 1741 svqvseanli gtgtsehwvd degpqpkkvk aedeallseeddpidrrpwt qqhleaadir 1801 rtpslaltpp qaeqevdvld vnvrgpdgct plmlaslrggssdlsdeded aedssaniit 1861 dlvyqgaslq aqtdrtgema lhlaarysra daakrlldagadanaqdnmg rcplhaavaa 1921 daqgvfqili rnrvtdldar mndgttplil aarlavegmvaelincqadv navddhgksa 1981 lhwaaavnnv eatllllkng anrdmqdnke etplflaaregsyeaakill dhfanrditd 2041 hmdrlprdva rdrmhhdivr lldeynvtps ppgtvltsalspvicgpnrs flslkhtpmg 2101 kksrrpsaks tmptslpnla keakdakgsr rkkslsekvqlsessvtlsp vdslesphty 2161 vsdttsspmi tspgilqasp npmlataapp apvhaqhalsfsnlhemqpl ahgastvlps 2221 vsqllshhhi vspgsgsags lsrlhpvpvp adwmnrmevnetqynemfgm vlapaegthp 2281 giapqsrppe gkhittprep lppivtfqli pkgsiaqpagapqpqstcpp avagplptmy 2341 qipemarlps vafptammpq qdgqvaqtil payhpfpasvgkyptppsqh syassnaaer 2401 tpshsghlqg ehpyltpspe spdqwssssp hsasdwsdvttsptpggagg gqrgpgthms 2461 epphnnmqvy aAssay Methods

As described above, the skilled person is well aware of numerous assayformats which may be appropriate for determining interaction betweenNotch and NB-3 and/or F3, and identifying substances which modulate,preferably promote, such interaction.

For example, interaction between the proteins may be studied in vitro bylabelling one or more with a detectable label and bringing it intocontact with another which has been immobilised on a solid support.Suitable detectable labels, especially for petidyl substances include³⁵S-methionine which may be incorporated into recombinantly producedpeptides and polypeptides. Alternatively the complex formed on the solidsupport may be detected by labelling with an antibody directed againstan epitope present on a protein which is not immobilised on the solidsupport. If no suitable antibody is available, a recombinantly-producedpeptide or polypeptide may be expressed as a fusion protein containingan epitope against which a suitable antibody is available.

The protein which is immobilized on a solid support may be immobilizedusing an antibody against that protein bound to a solid support or viaother technologies which are known per se.

A preferred in vitro interaction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described above atest compound can be assayed by determining its ability to affect theamount of labelled peptide or polypeptide which binds to the immobilizedGST-fusion polypeptide. This may be determined by fractionating theglutathione-agarose beads by SDS-polyacrylamide gel electrophoresis.Alternatively, the beads may be rinsed to remove unbound protein and theamount of protein which has bound can be determined by counting theamount of label present in, for example, a suitable scintillationcounter.

An assay according to the present invention may also take the form of acell-based assay in which at least one of the proteins is expressed by,preferably on the surface of, a suitable cell. The assay may utilise acell line, such as a yeast strain or mammalian cell line, in which therelevant polypeptides or peptides are expressed from one or more vectorsintroduced into the cell.

Modulators of Notch/NB-3/F3 interaction identified by the methodsdescribed may be further modified to increase their suitability for invivo administration.

Formulations

The compositions of the invention may be prepared as pharmaceuticalformulations comprising at least one active compound, as defined above,together with one or more other pharmaceutically acceptable ingredientswell known to those skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, adjuvants, excipients, buffers,preservatives and stabilisers. The formulation may further compriseother active agents.

Thus, the present invention further provides a method of making apharmaceutical composition as previously defined, the method comprisingadmixing at least one active agent as described herein together with oneor more pharmaceutically acceptable ingredients well known to thoseskilled in the art, e.g., carriers, adjuvants, excipients, etc.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, adjuvant, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

Suitable carriers, adjuvants, excipients, etc. can be found in standardpharmaceutical texts, for example Remington's Pharmaceutical Sciences,20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook ofPharmaceutical Excipients, 2nd edition, 1994.

Formulations may suitably be injectable formulations, e.g. in the formof aqueous, isotonic, pyrogen-free, sterile solutions, in which theactive compound is dissolved. Such liquids may additional contain otherpharmaceutically acceptable ingredients, such as anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, suspending agents, thickeningagents, and solutes which render the formulation isotonic with the bloodor cerebrospinal fluid. Examples of suitable isotonic carriers for usein such formulations include Sodium Chloride Injection, Ringer'sSolution, or Lactated Ringer's Injection. Typically, the concentrationof the active compound in the liquid is from about 1 ng/ml to about 10μg/ml, for example from about 10 ng/ml to about 1 μg/ml. Theformulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, and may be stored in afreeze-dried (lyophilised) condition requiring only the addition of thesterile liquid carrier, for example water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules, and tablets.

Administration

Administration of the compositions of the invention will generally be byinjection, preferably directly into the CNS. Injection may be directlyinto the site of damage. Alternatively, injection may be into thecerebro-spinal fluid, typically near the site of injury or illness.

Sequence Identity

Percent (%) amino acid sequence identity with respect to a referencesequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe reference sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. % identity values may be determined by WU-BLAST-2(Altschul et al., Methods in Enzymology, 266:460-480 (1996)). WU-BLAST-2uses several search parameters, most of which are set to the defaultvalues. The adjustable parameters are set with the following values:overlap span=1, overlap fraction=0.125, word threshold (T)=11. A % aminoacid sequence identity value is determined by the number of matchingidentical residues as determined by WU-BLAST-2, divided by the totalnumber of residues of the reference sequence (gaps introduced byWU-BLAST-2 into the reference sequence to maximize the alignment scorebeing ignored), multiplied by 100.

Percent (%) amino acid similarity is defined in the same way asidentity, with the exception that residues scoring a positive value inthe BLOSUM62 matrix are counted. Thus, residues which are non-identicalbut which have similar properties (e.g. as a result of conservativesubstitutions) are also counted.

In a similar manner, percent (%) nucleic acid sequence identity withrespect to a reference nucleic acid is defined as the percentage ofnucleotide residues in a candidate sequence that are identical with thenucleotide residues in the reference nucleic acid sequence. The identityvalues used herein may be generated by the BLASTN module of WU-BLAST-2set to the default parameters, with overlap span and overlap fractionset to 1 and 0.125, respectively.

The Subject

The subject to which the compositions and/or treatments of the inventionwill be administered will be a mammal, preferably an experimental animalsuch as a rodent (e.g. a rabbit, rat or mouse), dog, cat, monkey or ape,or a farm animal such as a cow, horse, sheep, pig or goat. Morepreferably, the subject is human.

Generally, the subject will have CNS damage, usually resulting from adisease or disorder characterised by inadequate myelination. Suchconditions include MS. In experimental animals, the damage or disordermay be experimental. The CNS damage may also result from physical injurye.g. spinal cord injury (SCI) other diseases or disorders, e.g. stroke,epilepsy or a neurodegenerative condition, learning memory-relatedcondition and/or dementia such as Alzheimer's disease or Parkinson'sdisease.

The treatments of the invention may be used in conjunction with othertherapies, such as surgery and/or rehabilitation.

Mimetics

Non-peptide “small molecules” are often preferred to peptides orpolypeptides for in vivo pharmaceutical use. Accordingly, mimetics of F3and/or NB-3 and complexes thereof may be designed, especially forpharmaceutical use. Typically a mimetic of one or both of F3 and NB-3will be capable of binding to a Notch molecule, preferably theextracellular domain of Notch 1 or 2, to mimic the effects of thatprotein or proteins binding to the same molecule.

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable where the active compound is difficultor expensive to synthesise or where it is unsuitable for a particularmethod of administration, e.g. peptides are unsuitable active agents fororal compositions as they tend to be quickly degraded by proteases inthe alimentary canal. Mimetic design, synthesis and testing is generallyused to avoid randomly screening large number of molecules for a targetproperty.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g. by substituting each residue in turn. Alanine scans of peptide arecommonly used to refine such peptide motifs. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modelledaccording to its physical properties, eg stereochemistry, bonding, sizeand/or charge, using data from a range of sources, eg spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conveniently be selected so thatthe mimetic is easy to synthesise, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. Alternatively, where the mimetic ispeptide based, further stability can be achieved by cyclising thepeptide, increasing its rigidity. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimisation ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

In the present case, peptide mapping studies may be used to identify theminimal portion of either F3 or NB-3 required to interact with Notch.This peptide may then be used as a lead compound for mimetic design, asdescribed above.

Antibodies

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding substance having anbinding domain with the required specificity. Thus, this term coversantibody fragments, derivatives, functional equivalents and homologuesof antibodies, including any polypeptide comprising an immunoglobulinbinding domain, whether natural or synthetic. Chimaeric moleculescomprising an immunoglobulin binding domain, or equivalent, fused toanother polypeptide are therefore included. Cloning and expression ofchimaeric antibodies are described in EP-A-0120694 and EP-A-0125023.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston etal, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993).

Diabodies are multimers of polypeptides, each polypeptide comprising afirst domain comprising a binding region of an immunoglobulin lightchain and a second domain comprising a binding region of animmunoglobulin heavy chain, the two domains being linked (eg by apeptide linker) but unable to associate with each other to form anantigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804).

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449(1993)), eg prepared chemically or from hybrid hybridomas, or may be anyof the bispecific antibody fragments mentioned above. It may bepreferable to use scFv dimers or diabodies rather than whole antibodies.Diabodies and scFv can be constructed without an Fc region, using onlyvariable domains, potentially reducing the effects of anti-idiotypicreaction. Other forms of bispecific antibodies include the single chain“Janusins” described in Traunecker et al, Embo Journal, 10, 3655-3659,(1991).

It may be desirable to “humanise” non-human (eg murine) antibodies toprovide antibodies having the antigen binding properties of thenon-human antibody, while minimising the immunogenic response of theantibodies, eg when they are used in human therapy. Thus, humanisedantibodies comprise framework regions derived from human immunoglobulins(acceptor antibody) in which residues from one or more complementarydetermining regions (CDR's) are replaced by residues from CDR's of anon-human species (donor antibody) such as mouse, rat or rabbit antibodyhaving the desired properties, eg specificity, affinity or capacity.Some of the framework residues of the human antibody may also bereplaced by corresponding non-human residues, or by residues not presentin either donor or acceptor antibodies. These modifications are made tothe further refine and optimise the properties of the antibody.

Aspects and embodiments of the second aspect of the present inventionwill now be illustrated, by way of example, with reference to theaccompanying figures. Further aspects and embodiments will be apparentto those skilled in the art. All documents mentioned in this text areincorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES RELATING TO THE SECOND ASPECT OF THEPRESENT INVENTION

FIG. 1. Adhesion of OLN-93 cells or mouse Notch1 transfected Hela cellswith or without antibody blocking treatment to spots of proteins, suchas F3-Fc (a, k-l, and q), CHL1-Fc (a) and NB-3-His (b, e-j, m-p, and r),applied to nitrocellulose was determined. Bars (a-b and q-r) representthe number of adherent cells (mean±SD) from at least three independentexperiments. Bar marked * is highly significantly (P<0.05) differentfrom the control.

Adhesion of OLN-93 cells on coated F3-Fc and NB-3-His substrates. a.Effect of antibody blockade on OLN-93 cell adhesion to F3-Fc substrate.F3: F3 protein substrate only (as a control); CHL1: CHL1 proteinsubstrate only; Anti-F3 or Anti-NB-3: addition of these two antibodies,respectively, to block the F3-Fc coated substrates before plating OLN-93cells; Anti-Notch1, Anti-Notch2 or Serum: pre-treated OLN-93 cells withthese two specific blocking antibodies or pre-immune serum respectivelybefore plating upon a F3-Fc coated substrate.

b. Effect of antibody blockade on OLN-93 cell adhesion to NB-3substrate. NB-3: NB-3.His protein substrate only (as control); Anti-NB-3or Anti-F3: addition of these two antibodies, respectively, to block theNB-3-His coated substrates before plating OLN-93 cells; Anti-Notch1,Anti-Notch2 or Serum: pre-treated OLN-93 cells with these two specificblocking antibodies or pre-immune serum respectively before plating upona NB-3-His coated substrate.

c-d: Immunofluorescence micrographs of OLN-93 cells stained usinganti-Notch1 antibody (c) and anti-Notch2 antibody (d). Cell surfacestaining is present in both instances.

e-j: Bright-field micrographs of OLN-93 cells upon contact with coatedNB-3-His substrate after 0.5 hour in culture (e) and in the presence ofblocking antibodies against Notch1 (f), NB-3 (g), Notch2 (h), F3 (i),and pre-immune serum (j). Scale bar in (j): 8 μm for (c-j).

Adhesion of mouse Notch1 transfected Hela cells on coated F3-Fc andNB-3-His substrates.

k-p: Bright-field micrographs of mock-transfected Hela cells (k) andmouse Notch1 transfected Hela cells (1-p) upon contact with F3-Fc (k-1)or NB-3-His (m-p) and in the presence of blocking antibodies againstNB-3 (n), Notch1 (O), and pre-immune serum (p). Scale bar in (p): 8 μmfor (k-p).

q. Effect of antibody blockade on mouse Notch1 transfected Hela cellsinteraction with F3-Fc substrate. F3: F3-Fc coated substrate only;Anti-F3: addition of these antibodies to block the F3-Fc coatedsubstrates before plating mouse Notch1 transfected Hela cells;Anti-Notch1 or Serum: pre-treated mouse Notch1 transfected Hela cellswith these specific blocking antibodies or pre-immune serum respectivelybefore plating upon a F3-Fc coated substrate; Hela cells: platingmock-transfected Hela cells upon a F3-Fc coated substrate (as acontrol).

r. Effect of antibody blockade on mouse Notch1 transfected Hela cellsadhesion to NB-3-His coated substrate. NB-3: NB-3.His coated substrateonly; Anti-NB-3: addition of these antibodies to block the NB-3.Hiscoated substrates before plating mouse Notch1 transfected Hela cells;Anti-Notch1 or Serum: pre-treated mouse Notch1 transfected Hela cellswith these specific blocking antibodies or pre-immune serum respectivelybefore plating upon a NB-3.His coated substrate; Hela cells: platingmock-transfected Hela cells upon a NB-3.His coated substrate (as acontrol).

FIG. 2. Biochemical and cellular analysis of the interaction betweenNotch and F3/NB-3.

a-d: Reciprocal association of both F3 and NB-3 with Notch1 and Notch2.Lysates of rat brain were analyzed by co-immunoprecipitation with thesefour antibodies and beads and nonimmune IgG (as controls). In each case,lanes correspond to antibodies as marked (Anti-N1: Anti-Notch1 antibody,Anti-N2: Anti-Notch2 antibody). Western blots were probed withantibodies against F3 (a), NB-3 (b), Notch1 (c) and Notch2 (d).

e: Schematic diagram of the Notch1 molecule showing the terminologyassigned to each subcloned fragment.

f-g: Coomassie Brilliant blue staining (f) and immunoblot analysis (g)of the four fragments are shown. The anti-Notch1 antibody specificallyrecognizes N1.3 and N1.4.

h-i: Analysis of the interaction between Notch1 fragments with F3/NB-3by using a pull down assay. Rat brain lysates were incubated with GST orGST-fusion proteins (N1.1, N1.2, N1.3, N1.4) bound to Sepharose 4Bbeads. Bound proteins were eluted with SDS sample buffer and analysed bySDS-PAGE and Western blotting with antibodies against F3 or NB-3.

j-l: Adhesion of F3-transfected CHO cells (j), NB-3-transfected CHOcells (k), and mock-tranfected CHO cells (1) to four different Notch1fragments and GST. The protein fragments N1.1, N1.2, N1.3 and N1.4,together with GST as control were coated onto surfaces of petri dishesand F3-transfected CHO cells (j), NB-3-transfected CHO cells (k) andmock-transfected CHO cells (1) were plated and maintained in chemicallydefined medium for 2 hours. Bars represent the number of adherent cells(mean±SD) from at least three independent experiments. Bar marked * ishighly significantly (P<0.05) different from the control (GST)

FIG. 3. Western blot analysis of expression of MAG and PLP in co-cultureof F3-transfected CHO cells and OLN-93 cells.

a: Immunoblot analysis of MAG in rat brain homogenates (BRAIN) and cellco-culture extracts. b: Immunoblot analysis of PLP in cell co-cultureextracts. OLN: OLN-93 cell culture only; F3/OLN: co-culture of OLN-93cells and F3-transfected CHO cells; CHO/OLN: co-culture of OLN-93 cellsand mock-transfected CHO cells; TAG/OLN: co-culture of OLN-93 cells andTAG-1-transfected CHO cells; TAX/OLN: co-culture of OLN-93 cells andTAX-transfected CHO cells.

FIG. 4. Immunofluorescence localization of Jagged1 and NB-3.

a-c: At P2, hardly any NB-3 staining was detectable. Jagged1 stainingwas present as linear streaks consistent with an axonal localization.Scale bar in a-c: 20 μm for a-i.

d-f: At P5, NB-3 can be observed to cluster at paranodal locations.Significantly, there is a distinct boundary between Jagged1 and NB-3immunofluorescence, best seen in the enlarged images j-o. Scale bar inj-l: 2 μm for j-o.

g-i: At P14, the distribution of NB-3 and Jagged1 remains unchanged fromthe P5 pattern, apart from the fact that axon density and hence numbersof paranodes have increased.

FIG. 5. The schematic diagram of the molecular constituents of theparanode in the central nervous system.

At this location, multiple oligodendroglial cytoplasmic loops (herepictured only as a single loop) intimately contact the axolemma. Thepresent study has revealed that in addition to axonal F3/contactin andCaspr and glial neurofascin 155 (NF-155), other members of the axoglialjunction include axonal NB-3 and glial Notch. It has also beendemonstrated that a functional signalling interaction exists betweenF3/NB-3 and Notch. (N: Node of Ranvier, PN: Paranode, JPN:Juxtaparanode).

Table I

Primary rat oligodendrocytes were plated on coated F3-Fc, NB-3-His orBSA (control) substrates, respectively. After 2 hours in culture, totalRNA was extracted and subjected to real time RT-PCR analysis of MAG andPLP mRNA expression levels. Relative expression levels were derivedusing the comparative CT method. (CT: cycle threshold).

FIG. 6. Notch and F3 are binding partners.

(A) Cell adhesion assay. OLN cells were labeled with α-Notch1 (a) orα-Notch2 (b). OLN cells were plated on dishes spotted with F3 (c, e-i)or CHL1 (d). Cells were untreated or pre-treated prior to plating withα-Notch1 (e) or α-Notch2 (f), pre-immune serum (serum) (g), or withantigen-depleted α-Notch1 (D-α-Notch1) (h) or α-Notch2 (D-α-Notch2) (i).Dotted lines depict the edges of the protein-Fc spots. Adherent cellswere visualized by staining with Coomassie Blue. j: Quantification ofOLN cell adherence to F3 substrate and the effects of blockingantibodies. # p<0.05 compared with CHL1, * p<0.05 compared withpre-immune serum. Scale bar in (i): 20 μm for a, b; 120 μm for c-i.

(B) Cell repulsion assay. mN1-transfected HeLa cells (a) ormock-transfected HeLa cells (b) were plated on F3 coated dishes.Adherent cells were stained with Coomassie Blue. c: Quantification ofHeLa cell adherence to F3 and the effects of blocking antibodies. Insome experiments, mN1-transfected HeLa cells were pretreated with α-F3or α-Notch1, or with pre-immune serum (serum). # p<0.05 compared withmock-transfected HeLa cells, * p<0.05 compared with pre-immune serum.Scale bar in (b): 15 μm for a, b. Bar graphs (Aj, Bc) represent thenumber of adherent cells (mean±SD).

FIG. 7. Notch and F3 associate as a protein complex.

(A) F3 co-immunoprecipitates with Notch1 or Notch2. a:Immunoprecipitates from rat brain lysate were prepared using α-Notch1,α-Notch2, non-immune IgG or unconjugated beads, and were probed withα-F3. b: Reciprocal assays used OL-F3 to capture the protein complex,followed by immunoblotting with α-Notch1 or α-Notch2 to detect thebinding partner.

(B) Subcloning of the Notch1 extracellular domain. a: Schematic diagramof Notch1 and its subcloned fragments. b, c: Coomassie Blue staining andα-Notch1 immunoblot of the four fragments, respectively.

(C) F3 binds to specific domains of Notch1. a: The GST-Notch1extracellular fragments (N1.1, N1.2, N1.3 and N1.4) or GST alone wereused in a GST pull-down assay with rat brain lysate. The precipitatesand rat brain lysate (right lane) were probed for F3. b: Quantificationof mock- and F3-transfected CHO cells adhering to culture dishes coatedwith the four GST fusion fragments or GST alone. Bars represent thenumber of adherent cells (mean±SD). * p<0.05 compared with GST.

(D) Lipid raft analysis. F3 was mainly localized to the fifth fractionwhile Notch1 was enriched in fractions 9-12. Caspr was used as apositive control to mark lipid raft fractions. H: Total homogenate.

FIG. 8. NICD translocation.

(A) F3-induced NICD nuclear translocation. mNotch1-myc transfected OLNcells were treated with 11.2 nM F3 (a), Jagged1 (b), BSA (c) orpre-incubated with α-Notch1 EGF (11-12) prior to F3 treatment (d) andthen stained with α-NICD. e: Quantification of cells with nuclearstaining of NICD after treatment with increasing concentrations of F3and Jagged1. Data are mean±SEM. f: OLN cells were cultured withJagged1-, F3- or mock-transfected CHO cells, and lysates wereimmunoprobed with α-Notch1, α-Notch2 and α-tubulin.

(B) F3-induced RIP was carried out by γ-secretase at the S3 site. OLNcells were pre-treated with 200 μM γ-secretase inhibitor, stimulatedwith F3 (a) or Jagged1 (b), and stained with α-NICD. After treatmentwith F3 (c, d) or Jagged1 (e, f), myc-tagged V1744K or V1744Lmutant-transfected OLN cells were immunolabeled for c-myc. g: α-c-mycimmunoprecipitates from OLN cells expressing myc-tagged wild-type Notch1or V1744K and V1744L mutants were immunoblotted with α-NICD (whichrecognizes both the ˜300 kDa full-length and the ˜120 kDa intracellularportion of Notch1) or α-V1744 (which only recognizes NICD after cleavageat the S3 site). Scale bar in (Bf): 15 μm for Aa-d, Ba-f.

(C) Upregulation of Notch1 and Notch2. Total (cytoplasmic plus nuclear)NICD staining intensity was quantified in F3-treated and BSA-treatedmNotch1-myc transfected OLN cells (a). OLN cells cultured alone or withF3-, mock, TAG-1-, or TAX-transfected CHO cells were lysed and probedwith α-Notch1, o-Notch2 and α-Notch3 (b). c: Real-time PCR assay ofNotch mRNA levels in OLN cells treated with 11.2 nM F3, Jagged1 or PBS.Notch mRNA levels were normalized to β-actin. Bars are mean±SEM. *p<0.05compared with PBS.

FIG. 9. OLN cellular processes halt and alter their morphology uponcontact with F3-transfected CHO cells.

Cellular processes (arrows in a and b) of OLN cells extend towardsF3-transfected CHO cell somata and upon contact with them, terminate andelaborate a flattened cytoplasmic sheet that envelops the cell body.This phenomenon is indicated by asterisks (*). a: Both OLN andF3-transfected CHO cells were pre-stained with PKH26 red fluorescentdye. b: The bright-field micrograph corresponding to (a). c, f, i: OLNcells were pre-stained with PKH26 red fluorescent dye. d, g, j: Both OLNcells and F3transfected CHO cells were stained for c-myc (green). e, h,k: Merged images of (c, d), (f, g), (i, j), respectively. l-q: In thecontrol systems, cellular processes (arrowheads) of OLN cells extendpast transfected CHO cell bodies. (l, m), (n, o) and (p, q) arecorresponding PKH26 red fluorescent and bright-field micrographs ofco-cultures of OLN cells with mock-(l, m), TAX-(n, o) and TAG-1- (p, q)transfected CHO cells. r: Quantification of OLN cellular processesextending past transfected CHO cell bodies in the co-cultures. Data aremean±SD. * p<0.01 compared with controls. Scale bar in (q): 25 μm for(a,b,l-q) and 15 μm for (c-k).

FIG. 10. MAG is upregulated by F3/Notch interaction.

(A) MAG is upregulated by F3. CHO cells and transfected derivatives donot express classic ligands of Notch, Delta, Jagged1 and Jagged2 (a).Lysates of rat brain, OLN alone, and the indicated co-cultured cellswere probed with α-MAG (upper panel) or α-γ-tubulin (bottom panel) (b).(c) Measured by real-time PCR, MAG mRNA in primary OLs is elevatedsignificantly by F3, versus BSA treatment. The raw data were normalizedto GAPDH using comparative C_(T) method.

(B) F3, but not Jagged1, upregulates MAG. mNotch1-myc transfected OLNcells were treated with 11.2 nM F3-Fc (a, d) or Jagged1 (b, c, e, f) andlabeled using α-MAG (a, b) or α-CNPase (d, e). The arrows in (b, e)indicate the cell bodies, which can be better viewed in bright-fieldpictures (c, f). g: Fluorescence intensities of MAG and CNPase stainingin cells treated with F3, Jagged1, BSA, or pretreated with α-Notch1 EGF(11-12) followed by F3. Data are mean±SEM. h: Quantification of thesurface area (mean±SEM) occupied by cells treated with F3, Jagged1 orBSA. *p<0.05; **p<0.01 compared with BSA.

(C) MAG upregulation is F3/Notch interaction-dependent.

a-j: OLN cells transfected with Notch ICD-deleted mutants, dn-N1 (a),dn-N2 (b); LacZ (O); S3 cleavage mutants, V1744K (d) and V1744L (e);Notch ECD-deleted mutants, caN1 (f, g) and caN2 (h, i), were treated(a-e) or untreated (f-i) with 11.2 nM F3 and double labeled for MAG(red) and V5 (a-c, f, h) (green) or c-myc (d, e) (green). Thetransfected cells in f, h can be better viewed as indicated by arrows inbright-field pictures g, i, respectively. j: MAG fluorescenceintensities in cells transfected with various indicated constructsfollowed by different protein treatments. Data are mean i SEM. ECD:extracellular domain; TM: transmembrane domain: ICD: intracellulardomain. The S3 site mutations in V1744K and V1744L constructs wereindicated by triangles in the transmembrane region. * p<0.01 comparedwith F3-treated OLN cells. Scale bar in (Ci): 20 μm for (Ba-f); 40 μmfor (Ca-i).

FIG. 11. MAG expression is independent of Hes1 and dependent on DTX1.

(A) MAG upregulation is independent of Hes1 expression. (a) OLN cellswere treated with the indicated ligands or compounds. At the timesshown, Hes1 transcripts were quantified using real-time PCR andnormalized to that at the start of the time course. (b, d, e) OLN cellswere untreated or pre-treated with Hes1 sense (Hes1-S) or antisense(Hes1-AS) oligonucleotides followed by 11.2 nM F3. Cell lysates wereprobed with a-Hes1 (upper panel) or a-nuclear matrix protein (N-matrix)(bottom panel) (b) or cells were labeled for MAG (d, e). Also, OLN cellstransfected with pGVB/Hes1 reporter alone or together with constructsexpressing caN1, RBP-J, or myc-tagged dn-RBP-J were subjected toluciferase assay (c). Data are mean±SD. f: OLN cells transfected withdn-RBP-J-myc were treated with 11.2 nM F3 and double stained for MAG(red) and c-myc (green). g: MAG staining intensity in OLN cells withvarious treatments indicated above. Data are mean±SEM. *p<0.01 comparedwith cells treated with F3 alone.

(B) MAG upregulation involves DTX1. a: DTX1 constructs used inluciferase reporter assays and immunostaining study. N terminal: Nterminal domain; Pro: Proline-rich motif; Ring finger: Ring-H2 fingermotif. OLN cells were transfected with pGVB/Hes1 reporter alone ortogether with indicated expression constructs. b: Luciferase reporteractivity in these cells. Data are mean±SD.

DTX1 (c-e)-, DTX1-D1-HA (f-h)-, or DTX1-D2-Flag (i-k)-transfected OLNcells were treated with 11.2 nM F3 and double labeled for MAG (red) andrelated tags (green). 1: MAG fluorescence intensity in OLN cellstransfected with indicated constructs followed by different proteintreatments. Data are mean±SEM. * p<0.01 compared with F3-treated OLNcells. Scale bar in (Bk): 30 μm for Ac-f, Bc-k.

FIG. 12. F3/Notch signaling via DTX1 promotes OPC differentiation.

Purified Ng2+/CNPase-OPCs (a), were treated with BSA (b), F3 (c) orJagged1 (d) for 2 days, double labeled for Ng2 (red) and CNPase (green)and counted (k). OPCs were also transfected with tagged dn-N1 (e, h) andDTX1-D2 (f, i), followed by F3 treatment or with caN1 and left untreated(g, j). Cells were double stained for the appropriate tag (green) andCNPase (red; e-g) or Ng2 (red; h-j), and counted (k). Scale bar in (j):25 μm for (a, e-inset), 100 μm for (b-j).

FIG. 13. Proposed model of distinct ligand-dependent Notch signalingpathways during development.

F3 interacts with the Notch receptor on the opposing cell surface tostimulate Notch/RBP-J signaling pathway to recruit DTX1 before or afterreleasing NICD into the cytoplasm. The NICD/RBP-J/DTX1 complex mayundergo specific but unidentified modification prior to translocationinto the nucleus where it activates target genes such as MAG. Thissignaling may contribute to OL maturation after P6 when decreasedJagged1 expression favors the initiation of F3/Notch signaling. Incontrast, before P6, Jagged1/Notch signaling activates theNICD/RBP-J-dependent transcription of target genes such as Hes1 andpredominantly inhibits OPC differentiation. ECM: Extracellular matrix;C: Cytoplasm; N: Nucleus; NICD^(Jag1), NICD^(F3): NICD released uponJagged1 and F3 activation, respectively; E: embryo; P6, P15: postnatalday 6 and 15, respectively; A: adult; OPC: oligodendrocyte precursorcell; O: oligodendrocyte; right bottom cartoon: myelinatingoligodendrocyte ensheathing the axon.

FIG. 14. NB-3 is a paranodal neuronal molecule.

A. NB-3 is expressed by neurons. Purified neurons, OLs and astrocytes ofE17 rats were double stained for NB-3 and corresponding surface marker:NF200 (a), Gal-C (b) and GFAP (c), respectively. Scale bar in (c): 30 μmfor (a-c).

(d) NB-3 is expressed from E17. Brain stem from rats with indicated ageswere homogenized and subjected to immunoblot for NB-3, F3, MAG.

(B) NB-3 is localized at the paranode. Brain stem sections from 90 dayold rats were double labeled for NB-3 and Caspr (a-c) or NB-3 and sodiumchannels (d-f). Scale bar in (f): 15 μm for (a-f).

(C) Lipid raft assay. NB3 was enriched in fraction 5, the same fractionas F3 and Caspr. H: Total homogenate.

FIG. 15. NB-3 is a functional ligand of Notch1.

(A) NB-3 binds to Notch1. (a) NB-3 co-immunoprecipitated with Notch1.Immunoprecipitates from rat brain lysates using α-Notch1 and α-NB-3 wereprobed with α-NB-3 or α-Notch1, respectively. (b) Cell adhesion assay.OLN cells were seeded on NB-3 substrate and adhere to it. Adhesion wasspecifically blocked by α-Notch1 or α-NB-3. # p<0.05 compared withCHL1; * p<0.05 compared with pre-immune serum. (c) NB-3 binds tospecific region on Notch1. The Notch1 GST fusion proteins or GST alonewere used in a GST pull-down assay from rat brain lysates. Theprecipitates and brain lysates were probed for NB-3. (d) Quantificationof adherent NB-3- and mock-transfected CHO cells to the four Notch1 GSTfusion fragments. * p<0.05 compared with GST. Bar graphs (b, d)represent the number of adherent cells (mean±SD).

(B) NB-3/Notch interaction induces NICD nuclear translocation in OLNcells. mNotch1-myc transfected OLN cells treated with NB-3 (a), Jagged1(b) and BSA (c) were immunostained for NICD. Some cells were treatedwith EGF antibody (d) or γ-secretase inhibitor (e) before NB-3stimulation. OLN cells were also transfected with V1744K-myc (f, h) orV1744L-myc (g, i), treated with NB-3 (f, g) or Jagged1 (h, i), andimmunostained with c-myc antibody to locate NICD. Scale bar in (a): 20μm for (a-i). (j) After NB-3 or Jagged1 treatment, α-c-myc precipitatesfrom mNotch1-myc, V1744K-myc or V1744L-myc transfected OLN cells wereimmunoblotted by α-NICD (upper panel) or α-V1744 (lower panel).

(C) Hes1 and Hes5 are not activated by NB-3. OLN cells treated with NB-3for different durations as indicated were lysed and the extracted mRNAsubjected to real-time PCR (a). The data were normalized to the mRNAlevel at the starting point. Other cells were treated with PBS, BSA, L1or NB-3 for 48 hours and analysed by real-time PCR (b). (c) OLN cellswere transfected with Hes1 or Hes5 luciferase reporter alone followed byNB-3 treatment or with caN1 construct. 24 hours post-transfection, cellswere subjected to luciferase assay. Data are mean±SD.

FIG. 16. NB-3/Notch interaction upregulates MAG via DTX1.

(A) MAG was upregulated in the co-culture of OLN-93 cells andNB-3-transfected CHO cells (a). N-matrix: nuclear matrix protein.CHO/OLN, NB3/OLN: co-culture of OLN-93 cells and mock- orNB-3-transfected CHO cells, respectively. (b) MAG mRNA in primary OLsincreased about 24 fold after NB-3 stimulation as monitored by real-timePCR. GAPDH was used as an internal control. OLN cells were treated withNB-3 (c), Jagged1 (d, e) and BSA (not shown) and immunostained for MAG.The fluorescence intensity of MAG was counted (f). Data are mean±SEM.

(B) NB-3-induced MAG upregulation involves DTX1. OLN cells weretransfected with dn-N-1-V5 (a), V1744K-myc (b), V1744L-myc (c), caN1 (d,e), dn-RBP-J-myc (h), DTX1-myc (i), DTX1-Dl-HA (j), and DTX1-D2-Flag(k), and treated with NB-3. The cells were then immunostained for MAGand corresponding tag. The fluorescence intensity of MAG was counted intransfected and non-transfected cells (1). Data are mean±SD. (f)Schematic structure of DTX1 and its two deletion mutants. Number 1, 2, 3correspond to N-terminal, proline-rich region and Ring-H2 finger motif,respectively. (g) Hes1 luciferase reporter assays to confirm thevalidity of indicated constructs. Data are mean±SD. The scale bar in(k): 30 μm for (Ac-e, Ba-k).

FIG. 17. NB-3 developmentally clustering at paranodes promotes OPCdifferentiation via Notch1/DTX1 signaling pathway.

(A) NB-3 and Jagged1 are distinctly distributed during development.Brain stems from P2 (a), P5 (b, c) rats were double stained for Jagged1(green) and NB-3 (red). The scale bar in (c): 30 μm for (a, b), 5 μm for(c).

(B) NB-3/Notch accelerates OPC differentiation via DTX1. Purified Ng2+OPCs from P7 rat optic nerve (a) were treated with BSA (b), NB-3 (c) orJagged1 (d) for 2 days and double labelled for Ng2 (red) and CNPase(green). Other cells were transfected with dn-N1 (e), DTX1-D1 (f)followed by NB-3 stimulation or caN1 (g) alone. Cells were thenimmunostained for tags (green) and CNPase (red). The percentage ofCNPase+cells were counted (h). Data are mean±SEM. The scale bar in (g):40 μm for (a-g).

FIG. 18. NSC: Rat brain stems of indicated ages were subjected toWestern blot for NB-3 and Notch1 expression patterns.

FIG. 19. NSC: NB-3 is a functional ligand of Notch1.

(A) NSCs express Notch1. NSCs were double stained for precursor markernestin (a) and Notch1 (b). (c) is the merged picture. Scale bar in (c):60 μm for (a-c).

(B) NB-3 binds to Notch1. P0 rat brain samples were precipitated byProtein A beads coupled with NB-3-Fc fusion protein, α-Notch1 or α-NB-3and the precipitates were blotted as indicated (a). N1-transfected Helacells (N1) were seeded onto coated NB-3 substrate in the absence (b) orpresence of blocking antibodies: α-NB-3 (c), α-Notch1 (d) or pre-immuneserum (s) (e). The adherent cells were counted (f). Data are mean±SD. *p<0.05 compared with mock-transfected Hela cells; # p<0.05 compared withpre-immune serum.

(C) NB-3 induces NICD nuclear translocation. NSCs were individuallytreated with NB-3(12.5 nM) (a), Jagged1(501M) (b) or BSA (c) for 24hours then fixed and triple stained for nestin (green), NICD (red) andHoechst 33258 (blue) to locate NICD. Scale bar in (c): 20 μm for (a-c).

(D) NB-3 does not activate Hes1. NSCs were transfected with Hes1luciferase reporter construct followed by NB-3 or Jagged1 treatment orcotransfected with caN1.24 hours post-transfection, the cells were lysedand subjected to luciferase assays (a). Other NSCs were transfected withpE7 luciferase reporter together with indicated constructs with orwithout NB-3 treatment and subjected to luciferase assays (b). Data aremean±SD.

FIG. 20. NSCs: NB-3 promotes OL generation.

NSCs were passaged into mitogen-withdrawn culture medium that wassupplemented with NB-3 (a, d), BSA (b, e) or Jagged1 (c, f). After 7 DIVdifferentiation, the cells were triple stained for CNPase (a-c, red) orβ-tubulin (d-f, red), GFAP (a-f, green) and Hoechst 33258 (a-f, blue).Other NSCs were individually immunolabelled with marker antibody andsubjected to flow cytometry. The percent of each type of cells: OLs,neurons and astrocytes were counted (g). Data are mean±SEM. Scale bar in(f): 40 μm for (a-f).

FIG. 21. NB-3/Notch signalling pathway via DTX1 instructsoligodendrogliogenesis.

NSCs were transfected with dn-N1 (a, b), DTX1-D2 (d, e) followed by NB-3treatment or caN1 (f, g) and double stained for appropriate tags andCNPase (a, d, f) or GFAP (b, e, g). (c) Schematic structure of DTX1 andDTX1-D2 constructs. The validity of the constructs utilized here wasconfirmed in Hes1 luciferase reporter assays (h). The percents oftransfected cells that were positive for CNPase or GFAP were counted(i). Data are mean±SD. Scale bar in (g): 20 μm for (a, b, d-g)

FIG. 22. The putative model.

The extracellular NB-3/Notch interaction releases from the membraneNICD, which recruits DTX1 and translocates into the nucleus where thecomplex mediates directly or indirectly CNPase expression, thuspromoting oligodendrogliogenesis. NSC: neural stem cell; O: OLs; M:membrane; N: nucleus; CNP: CNPase.

DETAILED DESCRIPTION OF THE SECOND ASPECT OF THE INVENTION

Results

Notch is the Oligodendroglial Surface Receptor for F3 and NB-3

The inventor set out to identify the glial receptor for F3 and NB-3.Both molecules have a basic structure composed of immunoglobulin andfibronectin type III repeats. Previous work investigated the effect ofF3/tenascin-R (TN-R) interaction in various in-vitro models (Xiao et al,1996, 1997, and 1998 all incorporated herein by reference). Of relevanceto the present study, the inventor had demonstrated that the epidermalgrowth factor-like repeats of TN-R constituted the binding site for itsneuronal receptor F3. The extracellular domain of Notch is composedprimarily of epidermal growth factor-like repeats, hence making it aplausible candidate as the glial receptor of F3 and NB-3. In addition tothis structural factor, the temporal and spatial location of Notch onthe maturing oligodendrocyte in contact with the axon additionallyattests to its suitability as a receptor for paranodal F3 and NB-3. Asthe experiments utilized the oligodendrocyte cell line OLN-93, it wasconfirmed using immunocytochemistry that these cells indeed express bothNotch1 and Notch2 on their surface (FIG. 1 c-d).

The inventor first carried out substrate adhesion assays to determine ifF3 and NB-3 could be binding partners of Notch. To do this, the OLN-93cells were cultured on F3 and NB-3 protein substrates in the presence orabsence of respective blocking antibodies against F3, NB-3 and Notch.The results show that OLN-93 cells adhered readily to F3 and that theadhesive effects were blocked by antibodies against the F3 substrate andboth Notch1 and Notch2 (FIG. 1 a-j). Cell adhesion to the F3 substratewas also reduced when NB-3 antibodies were added. The OLN-93 cells alsoadhered to NB-3 and incubation with antibodies against NB-3, Notch1 andNotch2 reduced cellular adhesion. In addition, when OLN-93 cells wereplated upon NB-3, they rapidly underwent a marked morphological change.They enlarged and transformed into oval-shaped to circular flattenedcells characterized most strikingly by an expansive cytoplasmic sheet(FIG. 1 c-j). This alteration in morphology also occurred when OLN-93cells were plated upon the F3 substrate, but only after a longerduration (not shown). As controls, the inventor used bovine serumalbumin and CHL1 (Holm et al, 1996), another neural cell adhesionmolecule of the immunoglobulin superfamily, but neither substratepromoted OLN-93 cell adhesion. This suggests that the F3/NB-3 signal mayconstitute a mechanism that triggers oligodendroctye differentiation.

Additional evidence for the interaction between F3/NB-3 and Notch wasprovided using the same assay system and blocking antibodies, but usingmouse Notch1-transfected HeLa cells instead of OLN-93 cells (FIG. 1k-r). In this instance, the inventor noted interestingly thatNotch1-transfected HeLa cells were repelled from F3-Fc and thisrepulsive effect was reversed when antibodies against F3 and Notch1 wereadded. In the case of NB-3-His, there was adhesion of theNotch1-transfected HeLa cells, which was inhibited when antibodiesagainst NB-3 and Notch1 were added. These cellular studies stronglysuggest that Notch is a receptor for F3 and NB-3.

F3 and NB-3 Bind to Distinct Sites on Notch

To analyse further the presence of an association between F3 and Notchand between NB-3 and Notch, the inventor carried out several biochemicaland molecular approaches (FIG. 2). Rat brain membrane preparations weresolubilized in 2% Triton X-100 and were immunoprecipitated withantibodies to Notch1 and Notch2. Western Blot analysis using anti-F3 andanti-NB-3 showed that the anti-Notch1 and 2 antibodies precipitatescontained both F3 and NB-3 (FIG. 2 a-b). Control immunoprecipitates withnon-immune IgG were negative for both F3 and NB-3. In the reverseco-immunoprecipitation experiment, anti-F3 and anti-NB-3 was used toimmunoprecipitate similar brain preparations and the blot probed withantibodies to Notch1 and Notch2 (FIG. 2 c-d). Theseco-immunoprecipitation results provide evidence that both F3/Notch andNB-3/Notch interactions may underlie the formation of protein complexesin the brain.

To allow the inventor to further characterize this interaction, theextracellular domain of Notch1 was arbitrarily divided into four equalsized overlapping 1.5 kb fragments (FIG. 2 e) and subcloned each of themin frame into PGEX-KG (Guan and Dixon, 1991) for GST fusion proteinproduction. The four protein fragments were expressed by induction oftransformed E. coli. The inventor's nomenclature for the four Notch1protein GST fusion protein fragments is N1.1, N1.2, N1.3 and N1.4,proceeding in a N- to C-terminal direction. Coomassie Blue staining andimmunoblots of the four fragments are shown (FIG. 2 f-g). The antibodyto Notch1 specifically recognized N1.3 and N1.4 (FIG. 2 g). Given theinventor's finding that the adhesion of OLN93 cells to F3 and NB-3 wereblocked by same Notch1 antibodies, F3 and NB-3 may at least have acommon binding site on either N1.3 or N1.4.

The inventor then carried out a GST pull-down assay to provide furtherbiochemical evidence that an association exists between Notch and bothF3 and NB-3. The four Notch1 GST fusion proteins were used to bind bothF3 and NB-3 in a rat brain lysate. Upon analysis by Western blotting,they discovered that F3 associated with two fragments—N1.1 and N1.3(FIG. 2 h) whilst NB-3 associated with N1.3 only (FIG. 2 i). Theseresults serve to refine the inventor's earlier data that F3 and NB-3 arebinding partners of Notch and that they have a common binding site onN1.3.

As an added confirmation of this biochemical interaction, the inventorused the four Notch1 protein fragments to carry out cell adhesion assaysin which F3-transfected CHO cells and NB-3-transfected CHO cells wereplated upon each individual fragment. The results support the findingsfrom the GST pull-down assay, in that the F3-transfected cells boundpredominantly to N1.1 and N1.3 (FIG. 2 j) and the NB-3-transfected cellsbound predominantly to N1.3 (FIG. 2 k). Altogether, these resultsprovide biochemical evidence to support the notion that Notch acts as areceptor for F3 and NB-3.

The Expression of MAG is Upregulated by the Interaction Between Notchand F3/NB-3

The above results substantiated the inventor's hypothesis that amolecular interaction occurs between paranodal F3 and NB-3 andoligodendroglial Notch as he has provided evidence of their physicalassociation and also their respective axonal (F3, NB-3) and glial(surface expression of Notch1 on oligodendrocytes) locations. Next, heexplored how this signalling event could be related to myelination. Asthe myelinating oligodendrocyte contacts and wraps the axons in amulti-layered spiral sheath, the protein components of the myelin sheathlogically become upregulated as the event progresses. Thus usingco-cultured OLN-93 cells and F3- or NB-3-transfected CHO cells, theinventor investigated the expression of myelin-specific proteins inthese cellular models of F3/NB-3-Notch interactions. Pure OLN-93cultures and co-cultures between OLN-93 cells and mock-, TAG- orTAX-transfected cells were used as controls. These cellular cultureswere homogenized to prepare membrane extracts and analyzed them byimmunoblotting to ascertain the levels of MAG (myelin-associatedglycoprotein) and PLP (proteolipid protein), components of the myelinsheath in the CNS. It was shown that when OLN-93 cells were co-culturedwith F3-transfected CHO cells or with NB-3-transfected CHO cells (notshown), MAG became specifically upregulated (FIG. 3). PLP levels,however, were the same in all the culture systems investigated. Thissuggested that the F3/Notch or NB-3/Notch interaction was active in thesetting of myelination. To provide further evidence to support thisfinding, the inventor proceeded to analyze if a similar alteration inmyelin-specific proteins occurred when primary oligodendrocytes wereemployed instead of the OLN-93 cell line. Therefore, primary cultures ofrat (postnatal day 1 or 2) oligodendrocytes were prepared and platedthem upon F3-Fc and NB-3-His fusion protein substrates. BSA (bovineserum albumin) was used as a control substrate. Again, this directcell-protein contact strived to simulate the contact between axonalligands and glia. Two hours after the cells were plated onto theproteins, total RNA was isolated from each of these interacting systemsand performed real-time RT-PCR assays to measure the mRNA expressionlevels of MAG and PLP. It was noticed that in both the systems whereprimary oligodendrocytes were plated upon F3 and NB-3, MAG expressionlevels were approximately 8-fold higher than in the control system whereBSA was used as a substrate (Table I). Message levels of PLP howeverwere not found to be elevated when compared with the control. Thesefindings are in agreement with the western blot results and furtherconfirm the presence of myelin-specific gene up-regulation arising as aresult of F3/Notch and NB-3/Notch signalling.

NB-3 and Jagged1 Localize to Distinct Axonal Domains

It has been explained how Jagged1 influences oligodendrocytedifferentiation via Notch (Wang et al, 1998). When the oligodendrocytecellular processes encounter F3 and NB-3 at the paranodes, a separateinstruction could be conveyed to the myelinating cell, nevertheless viathe same receptor—Notch. Having established that F3 and NB-3 areconfined to paranodal regions, it became apparent that if Jagged1 wereconfined to the axonal segment enclosed by paranodes, namely theinternode, a signal switch mechanism at work during axonal ensheathmentcould exist. The inventor therefore analyzed the distribution of NB-3and Jagged1 in rat brain stem sagittal cryosections usingimmunofluorescence (FIG. 4). Three separate age groups wereanalyzed—postnatal days 2, 5 and 14, thus allowing the axonal pattern ofNB-3 distribution to be investigated as the animal matured. Theinventors could not observe any staining for NB-3 at P2 (FIG. 4 a-c),whereas by P5 (FIG. 4 d-f), NB-3 could be seen clustered at paranodes.From this result, the inventors inferred that NB-3 is likely diffuselydistributed over the axonal surface initially and then translocates tothe paranodes during development to form distinct clusters. Importantly,Jagged1 immunoreactivity was confined to internodes and was separatefrom paranodes (FIG. 4 f). These findings suggest that the Notchreceptors on myelinating oligodendrocytes may switch axonal bindingpartners from Jagged1 to F3/NB-3 when they migrate along the axon fromthe internodes to adjacent paranodes, thus becoming sequentially exposedto different signals.

Notch is the Oligodendroglial Surface Binding Partner of F3.

Axonal F3 congregates at the paranode, a potential site for F3 tointeract with myelinating glia (Girault and Peles, 2002). Notch is aplausible binding partner since its extracellular portion possesses manyEGF-like repeats (Martinez Arias et al, 2002) and is abundantlyexpressed on maturing OLs (Lardelli et al, 1994; Wang et al, 1998). Toinvestigate this potential interaction an OL cell line OLN-93 (OLN) wasutilized. OLN cells were derived from spontaneously transformed cells inrat brain glial cultures and resemble maturing OLs (Richter-Landsbergand Heinrich, 1996). OLN cells no longer express the progenitor cellsurface marker A2B5, and are positive for only one isoform of myelinbasic protein (MBP) (˜14 kDa), characteristic of immature OLs.Immunocytochemistry confirmed that OLN cells express Notch1 and Notch2on their surface (FIG. 6Aa, b).

To investigate if F3 could bind to Notch, cell adhesion assays wereperformed as described (Xiao et al, 1996). OLN cells were plated onF3-Fc (F3) substrate in the absence or presence of blocking antibodies.OLN cells adhered readily to F3 (FIG. 6Ac), but not to CHL1-Fc (CHL1),another neural cell adhesion molecule (Holm et al, 1996) (FIG. 6Ad).Adhesion was blocked by pre-incubation with F3 (FIG. 6Aj), Notch1 orNotch2 antibodies (FIG. 6Ae, f, j), but not by pre-immune serum orantigen-depleted antibodies to Notch1 or Notch2 (FIG. 6Ag, h, i). MurineNotch1 (mN1)-transfected HeLa cells (Logeat et al, 1998) were also usedin cell repulsion assays, another approach to investigateligand-receptor relationships (FIG. 6B). mN1-transfected HeLa cells wererepelled from F3 (FIG. 6Ba) compared with mock-transfected HeLa cells(FIG. 6Bb). Repulsion was reversed by pre-treating the cells with F3 orNotch1 antibodies, but not with pre-immune serum (FIG. 6Bc). Thesestudies suggest that Notch1 interacts with F3.

F3 Binds to Specific Sites on Notch1

To confirm F3/Notch interaction, rat brain membrane samples wereimmunoprecipitated with Notch1 or Notch2 antibodies.

Immunoblotting of the precipitates using F3 antibody showed that theycontained F3 (FIG. 7Aa). In a reciprocal assay, an F3antibody-precipitate was probed with Notch1 or Notch2 antibodies (FIG.7Ab). These results indicate that Notch and F3 can form complexes.

To identify the specific site(s) on Notch1 for F3 binding the mouseNotch1 extracellular domain was divided into four equal-sized fragmentstermed N1.1, N1.2, N1.3 and N1.4 (FIG. 7Ba) and produced them asrecombinant GST fusion proteins as identified (FIG. 7Bb, c). The Notch1antibody used in the aforementioned cellular studies recognized N1.3 andN1.4, but not N1.1 or N1.2. GST pull-down assays with rat brain lysatesrevealed that F3 associated with N1.1 and N1.3 (FIG. 7Ca). To confirmthis, F3-transfected CHO cells (Gennarini et al, 1991) were seeded ontoculture dishes coated with the four GST fusion proteins. Cells boundpredominantly to N1.1 and N1.3. Mock-transfected CHO cells did not bind(FIG. 7Cb).

F3 and Notch1 are not Co-Localized in Lipid Rafts

F3 is a surface molecule localized in lipid rafts of OLs (Krämer et al,1999). To ascertain whether F3/Notch interaction could occur in cis inthese microdomains, they were isolated from P15 rat cerebral cortex.Both F3 and Caspr were detected in fraction 5 of the sucrose densitygradient (FIG. 7D) as reported (Faivre-Sarrailh et al, 2000). Notch1 wasfound only in fractions 9-12 that are enriched incytoskeleton-associated proteins (FIG. 7D). The same results wereobtained using adult rat cerebral cortex (not shown). Thus, F3 andNotch1 are unlikely to complex laterally within lipid rafts. Altogether,these observations suggest that F3 is a trans-binding partner of Notch.

NICD Translocates to the Nucleus After Notch Interacts with F3

The immediate consequence of Notch activation is the release andtransport of NICD to the nucleus (Schroeter et al, 1998). To determineif F3 acts as a functional ligand to initiate these events, myc-taggedfull-length mouse Notch1 (mNotch1-myc) was transfected into OLN cells.Cells were treated with different proteins and immunolabeled for NICDusing NICD antibody (Logeat et al, 1998). In F3-treated cellsconcentrated NICD staining was observed mainly in the nuclei (FIG. 8Aa),similar to Jagged1-induced NICD translocation (FIG. BAb). BSA failed totrigger this event (FIG. 8Ac). Pre-incubation with antibody to Notch1EGF repeats 11-12 (EGF 11-12 antibody, which crossreacts with Notch2,not shown) abolished F3-induced NICD translocation (FIG. 8Ad). BrefeldinA and monensin (not shown), compounds that inhibit the membraneinsertion of Notch1 (Schroeter et al, 1998), also prevented NICDtranslocation. Cell treatment with increasing concentrations of F3 orJagged1 led to a similar increase in nuclear clustering of NICD,indicating that translocation occurs in an F3 or Jagged1concentration-dependent manner (FIG. BAe). OLN co-cultured with eitherF3- or Jagged1-transfected CHO cells, but not with mock-transfected CHOcells, also resulted in production of Notch2 intracellular domain (ICD)(FIG. 8Af). Notch2 ICD antibody was not crossreactive with Notch1 ICD(not shown). These results demonstrate that F3, like Jagged1, canactivate Notch1 and Notch2, leading to subsequent nuclear translocationof NICD.

F3 Induces Notch Intramembrane Cleavage at the S3 Site.

As a prerequisite for activation, Notch undergoes RIP at the S3 site(V1744) by the presenilin-dependent γ-secretase (Schroeter et al, 1998;Huppert et al, 2000). To clarify the nature of F3-induced cleavage,mNotch1-myc transfected OLN cells were pre-incubated with γ-secretaseinhibitor and then treated with F3 or Jagged1. In both cases, no NICDstaining was observed in the nuclei (FIG. 8Ba, b). Moreover, two S3cleavage mutants: V1744K-myc and V1744L-myc, which showed reducedproteolysis and parallel reduction in activity (Schroeter et al., 1998),were transfected into OLN cells that were then treated with F3 orJagged1. Cells showed c-myc immunostaining mainly in the cytoplasm andon the cell surface, but not in the nuclei (FIG. 8Bc-f). Inimmunoprecipitation assays, c-myc antibody-precipitates from F3- orJagged1-treated V1744K-myc and V1744L-myc transfected OLN cells couldonly be labeled with NICD antibody that also recognizes full-lengthNotch1 (300 kDa) (FIG. 8Bg, upper panel), indicating that these mutantNotch1 molecules remained intact. Only the precipitates from F3- orJagged1-treated mNotch1-myc transfected OLN cells showed a reactive bandupon probing with V1744 antibody that solely recognizes NICD releasedfrom 53 (120 kDa) (FIG. 8Bg, lower panel). Altogether, theseobservations suggest that F3 induces RIP at the Notch1 S3 site.

F3/Notch Interaction Upregulates Notch1 and Notch2 Expression

F3, but not BSA, induced a two-fold increase in nuclear NICD (FIG. 8Ca),while there was no noticeable change in cytoplasmic NICD, suggestingthat F3 upregulates Notch expression. To investigate this, OLN cellswere cultured with mock-, F3-, TAG-1- or TAX-transfected CHO cells.TAG-1 and TAX are members of F3 subfamily (Tsiotra et al, 1993).Expression of Notch1 and Notch2, but not Notch3, increased when OLNcells were cultured with F3-transfected CHO cells (FIG. 8Cb). Real-timePCR confirmed that soluble F3 increased Notch1 and Notch2, but notNotch3 transcripts (FIG. 8Cc), while Jagged1 only upregulated Notch1,but not Notch2. Thus, F3/Notch interaction may provide a feedback loopto specifically upregulate Notch1 and Notch2.

Oligodendroglial Processes Alter their Morphology Upon Contact with F3

To model the scenario of axoglial contact during myelination, themorphology of OLN processes was studied when they contact cellsurface-expressed F3. Since OLN cells extend longer processes thanprimary OLs they are ideally suited for observing subtle morphologicalchanges that occur during contact. F3-transfected CHO cells mimicked theparanodal axonal component. Mock-, TAG-1and TAX-transfected CHO cellswere used as controls. Remarkably, most OLN processes terminated uponcontact with F3-transfected CHO cells and flattened to form acytoplasmic sheet spreading over the surface of CHO cells, as if in anattempt to envelop the cell (FIG. 9 a-k). But this was not observed withmock—(FIG. 91, m), TAG-1—(FIG. 9 n, o) or TAX-transfected (FIG. 9 p, q)CHO cells. While approximately 80% of extending processes halted uponreaching F3-transfected CHO cells, with other CHO cells the proportionwas only 20% (FIG. 9 r). These results show that a signal inducing themorphological change is presented to the oligodendroglial processes whenF3 is encountered.

F3, but Not Jagged1, Upregulates MAG

To explore how the morphological change described above could relate toF3/Notch signaling, the expression of myelin-associated glycoprotein(MAG) in the aforementioned co-cultures was investigated. Parental andtransfected CHO cells did not express Delta, Jagged1 and Jagged2 (FIG.10Aa). Membrane extracts of co-cultured cells were immunoblotted forMAG. The constitutive level of MAG in OLN cells was very low, if not,undetectable. However, when OLN cells were cultured with F3-transfectedCHO cells, MAG was upregulated (FIG. 10Ab). F3-transfected CHO cells donot express MAG (not shown). On the other hand, in real-time PCR,purified primary OLs plated upon F3 substrate showed approximatelysixteen-fold increase in MAG transcripts, versus cells seeded on BSA(FIG. 10Ac). OLN cells showed a similar efficiency of MAG upregulation(not shown).

In immunostaining assays, mNotch1-myc transfected OLN cells wereimmunolabeled for MAG and 2′, 3′-cyclic nucleotide 3′-phosphodiesterase(CNPase), an OLs specific antigen. Treatment with soluble F3 resulted ina remarkable increase in MAG staining (FIG. 10Ba) and enhanced CNPasestaining (FIG. 10Bd), compared with Jagged1 (FIG. 10Bb, c, e, f) or BSA(not shown) treatment. Quantification of MAG and CNPase fluorescenceintensities revealed an approximately 300% increase in MAG and 40%increase in CNPase labeling in F3- versus Jagged1- or BSA-treated cells(FIG. 10Bg). Cell pre-treatment with Notch 1 EGF 11-12 antibodyprevented F3-induced increase in MAG and CNPase (FIG. 10Bg), suggestingthat Notch is required for this event. In addition, F3 induced the cellsto flatten and form a sheet-like structure (FIG. 10Ba). Quantificationof the substratum area covered by cells revealed a two-fold increase inthat covered by F3-versus Jagged1- or BSA-treated cells (FIG. 10Bh).These findings confirm F3-induced MAG upregulation.

MAG is Upregulated by F3/Notch Interaction

To better understand the involvement of NICD in this event, OLN cellswere transiently transfected with V5-tagged dominant-negative Notch1(dn-N1) or Notch2 (dn-N2) (Small et al, 2001), which lack theintracellular regions but can bind to extracellular ligands. Cells werethen treated with F3 and double stained with V5 and MAG antibodies.Notably, both dn-N1-V5- (FIG. 10Ca) and dn-N-2-V5—(FIG. 10Cb) positivecells were poorly labeled for MAG. pcDNA4/V5/LacZ (LacZ) was used as avector control (FIG. 10Cc). Moreover, OLN cells transfected with two S3mutants, myc-tagged V1744K (FIG. 10Cd) and V1744L (FIG. 10Ce) weretreated with F3 and double stained for c-myc and MAG. In either case, F3failed to upregulate MAG in c-myc-positive cells. Quantification of MAGfluorescence intensity confirmed that Notch1 or Notch2 dysfunction, inother words, the absence of NICD, abolished F3-induced MAG upregulation(FIG. 10Cj), suggesting that NICD is required for MAG expression.

The inductive role of F3 in this event was further investigated byintroducing into OLN cells V5-tagged constitutive-active Notch1 (caN1)(FIG. 10Cf, g) and Notch2 (caN2) (FIG. 10Ch, i), which lack theextracellular domains and are ligand-independently active (Small et al,2001). That is, even in the absence of F3 stimulation, NICD is generatedand translocates to the nucleus. And in OLN cells, caN1 (FIG. 10Ac) andcaN2 (not shown) did lead to the transactivation of Hes1 in luciferasereporter assays. After transfection, cells were double stained for V5and MAG. Immunolabeling showed that V5-positive cells were faintlystained for MAG (FIG. 10Cj). Given that Jagged1 also induces NICDrelease, but does not increase MAG expression, these results demonstratethat MAG upregulation requires F3 induced NICD.

F3/Notch-Induced MAG Upregulation is Independent of Hes1

A prominent feature of Notch signaling is the activation of Hes genes inan oscillatory manner (Hirata et al, 2002). Hes1 mRNA expression in OLNcells was thus investigated. In real-time PCR, non-physiologicaltreatment of cells with 2 mM EDTA (Rand et al, 2000) triggered a sharpincrease in Hes1 mRNA during the first two hours and a return to basallevel by three hours (FIG. 11Aa), reflecting the exquisite intrinsicregulation of endogenous Hes1 expression (Dale et al, 2003). However,F3, Jagged1 or BSA treatment did not significantly alter the baselineoscillating levels of endogenous Hes1 transcription at short (the firstthree hours) or long (12 and 24 hours) times after treatment (FIG.11Aa).

To investigate whether F3-induced MAG upregulation is related toconstitutive levels of Hes1 protein, Hes1 antisense oligonucleotides(Kabos et al, 2002) were used to block basal Hes1 protein expression inOLN cells (FIG. 11Ab). MAG upregulation was not influenced by thistreatment or control sense oligonucleotides (FIG. 11Ad, e, g).

RBP-J is a Notch-regulated transcription factor that can activate Hesgenes (Martinez Arias et al, 2002). OLN cells were transfected withdominant-negative RBP-J-myc (dn-RBP-J-myc), bearing a mutation of lysine218 to histidine, which abolishes effective high affinity binding toHes1 promoter region (Kato et al, 1997). Hes1 luciferase reporter assayshowed that the mutant prevented Hes1 activation by caN1 (FIG. 11Ac).After transfection, cells were treated with F3 and double labeled forc-myc and MAG. c-myc positive cells showed the same level of MAG asneighboring nontransfected cells (FIG. 11Af, g). These results indicatethat MAG upregulation triggered by F3/Notch signaling is independent ofendogenous Hes1 expression.

F3/Notch-Induced MAG Upregulation Involves DTX1

Increasing evidence indicates that DTX1 is another downstream element ofthe Notch signaling pathway (Martinez Arias et al, 2002). Given thatF3-induced MAG upregulation is not related to Hes1 expression, the roleof DTX1 in this event was investigated using myc-tagged wild-type DTX1(Yamamoto et al, 2001) and its two deletion mutants as depicted (FIG.11Ba), namely, HA-tagged DTX1 mutant (DTX1-D1) containing amino acids1-411 (Yamamoto et al, 2001) and Flag-tagged DTX1 mutant (DTX1-D2)containing amino acids 1-242 (Izon et al, 2002). The two mutants lackthe Ring-H2 finger motif, which contributes to DTX1 oligomerization, anessential step for DTX1 functioning (Matsuno et al, 2002). As previouslyobserved (Yamamoto et al, 2001), Hes1 luciferase reporter assay showedoverexpressed DTX1 inhibited the transactivation of Hes1 by caN1 (FIG.11Bb). On the other hand, both DTX1-D1 and DTX1-D2 restored Hes1response to caN1 (FIG. 11Bb). After transfection, OLN cells were treatedwith F3 and double labeled for MAG and corresponding tags.Overexpression of DTX1 had no effect on F3-induced MAG upregulation(FIG. 11Bc-e, l). Interestingly, MAG upregulation was abolished in theHA-positive or Flag-positive cells (FIG. 11Bf-1). These observationsstrongly suggest that F3/Notch-induced MAG upregulation involves DTX1.

F3/Notch Signaling Pathway Via DTX1 Promotes OPC Differentiation IntoOLs

The Jagged1/Notch signaling pathway inhibits differentiation of OPCsinto OLs (Wang et al., 1998). To explore whether F3/Notch signaling viaDTX1 instructs OPC differentiation, purified OPCs positive for theprogenitor marker Ng2 (Dawson et al., 2000) (FIG. 12 a), were treatedwith BSA, F3 or Jagged1 for 2 days and then double stained for Ng2 andthe OL-specific CNPase (FIG. 12 b-d). After F3 treatment, over 70% ofOPCs differentiated into CNPase+ OLs compared to ˜50% after BSAstimulation, while Jagged1 treatment resulted in nearly all OPCsremaining undifferentiated (FIG. 12 k). F3-stimulated cells were morebifurcated and inclined to form a web-like structure (FIG. 12 c) thanBSA-treated cells (FIG. 12 b). Notch and DTX1 involvement was examinedby transfecting OPCs with dn-N1-V5 (FIG. 12 e, h) and DTX1-D2-Flag (FIG.12 f, i), respectively, followed by F3 treatment for 2 days. Other OPCswere transfected with caN1-V5 and left untreated (FIG. 12 g, j).Notably, immunolabeling for tags and CNPase (FIG. 12 e-g) or Ng2 (FIG.12 h-j) showed that most dn-N1 (˜75%) or DTX1-D2 (˜67%) transfected OPCsremained Ng2+ (FIG. 12 k), despite the presence of F3. In particular,40% of dn-N1-transfected cells were CNPase+, but these cells were lessbifurcated (FIG. 12 e, inset), compared to surrounding non-transfectedcells, indicating a relatively immature stage. However,CNPase+DTX-D2-transfected cells were hardly detectable. Consistent witha previous report (Wang et al., 1998), caN1-transfected cells remainedNg2+ and almost none became CNPase+ OLs (FIG. 12 k), indicating thatF3-induced NICD is specifically required for accelerated OPCdifferentiation. These results demonstrate that F3/Notch signaling viaDTX1 promotes OPC development.

NB-3 is Located at the Paranode

Central to the inventor's aims is the characterization of an interactionbetween an axonal ligand and an oligodendroglial receptor at theparanode, acting as a stop signal to prevent extending oligodendrocytecellular processes from impinging upon axonal domains destined to becomenodes of Ranvier. The first step was to demonstrate the identity of theaxonal ligands. Among the potential candidates, there is F3 which existsas a complex with Caspr at the paranode (R10s et al, 2000). Previousresults (Kazarinova-Noyes et al, 2001) are in agreement, showing that inrat optic nerve fibres, double-labelling with Caspr revealed thepresence of F3 in both paranodal and nodal locations.

Recently, NB-3, a GPI-linked cell adhesion molecule, has been identifiedas a member of F3/Contactin family (Lee et al, 2000). To determine thecell type(s) that express NB-3, purified primary neurons, OLs andastrocytes from E17 rat cerebella were separately cultured and doublestained for NB-3 and specific markers: neurofilament 200 (NF200) forneurons (FIG. 14Aa), galactocerebroside (Gal-C) for OLs (FIG. 14Ab) andglial fibrillary acidic protein (GFAP) for astrocytes (FIG. 14Ac). Theresults confirmed that only neurons expressed NB-3. NB-3 expression inrat brain stem development was next investigated by Western blot.Compared with F3, NB-3 expression started from E17, reached a plateaubetween P0 to P21, and declined afterwards (FIG. 14Ad), which parallelsthe time frame of OL development, as marked by increased expression ofMAG. These observations indicate that NB-3 is a neuron-derived molecule.These results suggest that NB-3/Notch signalling may play certain rolesin multiple phases of OL development. However, NB-3's exact localizationand physiological role has not been fully clarified.

F3/contactin and TAG-1, two members of the F3/contactin family, locatespecifically at nodal, paranodal, and juxtaparanodal regions (Giraultand Peles, 2002). In particular, the paranode flanks the node of Ranvierand forms the adhesive site for axoglial junctions, which is crucial forinitiating myelination and stabilization of myelin loops (Girault andPeles, 2002). To explore the role of NB-3 in axoglial interaction, NB-3distribution was analysed along myelinated axons. Doubleimmunofluorescence staining was performed by using monoclonal NB-3antibody, which does not cross-react with F3 (not shown), and polyclonalantibodies against Caspr or sodium channels on adult rat brain stemsagittal cryosections. Note that NB-3 overlapped exactly with Caspr(FIG. 14Ba-c) and flanked nodal sodium channels (FIG. 14Bd-f). Toconfirm this spatial specificity, NB-3 distribution in lipid rafts fromP15 and adult (not shown) rat cerebral cortex was studied. F3 and Casprare colocalized in lipid rafts. In agreement with the previous finding(Faivre-Sarrailh et al, 2000), these two paranodal molecules werecolocalized in fraction 5 of a sucrose density gradient. Interestingly,NB-3 was also mainly located in this fraction (FIG. 14C). Takentogether, these results indicate that NB-3 as a paranodal component mayco-localize with the F3/Caspr complex on myelinated axons.

NB-3 is a Functional Ligand of Notch1

Given its expression profile and specific location during OL maturation,the inventor was interested in finding whether NB-3 could interact withOL-derived Notch1. Immunoprecipitation assay showed that Notch1extracellular domain (˜190 kDa) was detected in a NB-3antibody-precipitate from adult rat brain membrane extracts while aNotch1 antibody-precipitate contained NB-3 (FIG. 15Aa), implying theexistence of NB-3/Notch complex. OLN-93 (OLN) cells, a permanent cellline resembling maturing Ols (Richter-Landsberg and Heinrich, 1996) wereused in cell adhesion assays. Immunocytochemistry showed that OLN cellsexpressed Notch1 on the surface (not shown). Cells adhered to the coatedNB-3 substrate, but not to CHL1, another neural cell adhesion molecule(Holm et al, 1996). Adhesion was blocked by pre-incubation with NB-3 orNotch1 antibodies, but not with F3 antibody or pre-immune serum (FIG.15Ab). The same results were obtained by using Notch1-transfected Helacells (not shown). To map the binding site(s) on Notch1, four subclonedsequential equal-sized portions of the mouse Notch1 extracellulardomain, labelled as N1.1, N1.2, N1.3, and N1.4 were used in a GSTpull-down assay from rat brain lysates. Immunoblotting showed that NB-3associated only with N1.3, a region containing EGF repeats 22-34 (FIG.15Ac). The specificity was confirmed by the observations thatNB-3-transfected CHO cells plated upon the individual recombinantfragments bound predominantly to N1.3 (FIG. 15Ad) while mock-transfectedCHO cells did not bind. Together, these results support the concept thatNB-3 is a ligand of Notch1.

NB-3/Notch Interaction Induces NICD Nuclear Translocation in OLN Cells

Upon binding its ligands, the core signalling mechanism of Notchinvolves Regulated Intramembrane Proteolysis (RIP) at the S3 site, whichreleases NICD into the nucleus (Schroeter et al, 1998). To explorewhether NB-3 is a functional ligand of Notch1, OLN cells transfectedwith myc-tagged full-length mouse Notch1 (mNotch1-myc) were treated withNB-3 (FIG. 15Ba), Jagged1 (FIG. 15Bb), or BSA (FIG. 15Bc) andimmunostained with NICD antibody (Logeat et al, 1998). Both NB-3 andJagged1, but not BSA, induced NICD to concentrate in the nucleus. Notch1EGF antibody targeting EGF-like repeats on Notch1 prevented NB-3-inducedNICD nuclear clustering (FIG. 15Bd). To study the properties ofNB-3-induced NICD release, OLN cells were pre-incubated with γ-secretaseinhibitor before exposure to NB-3 (FIG. 15Be) or Jagged1 (not shown). Inboth cases, nuclear NICD clustering was abolished, indicating thatNB-3-induced NICD release involves γ-secretase. Moreover, OLN cellstransfected with two S3 mutants: myc-tagged V1744K and V1744L whichabolished S3 cleavage (Schroeter et al, 1998), successfully preventedNB-3—(FIG. 15Bf, g) as well as Jagged1—(FIG. 15Bh, i) induced NICDnuclear translocation. In Western blot (FIG. 15Bj), c-myc antibodyprecipitates from either NB-3 or Jagged1-treated V1744K-myc orV1744L-myc-transfected OLN cells could only be blotted by NICD antibody,which also recognizes intact Notch1 (˜250 kDa) (upper panel), but not byV1744 antibody, which only recognizes released NICD from the S3 site. Incontrast, mNotch-myc transfected OLN cells generated NICD (˜120 kDa)that was blotted by V1744 antibody after NB-3 or Jagged1 treatment(lower panel). Together, these results demonstrated that NB-3 inducedγ-secretase-dependent Notch1 RIP at the S3 site.

Hes1 and Hes5 are not Activated by NB-3

In the nucleus NICD interacts with transcription factors, such as RBP-Jand/or Deltex1 (DTX1), thus activating target genes, such as HES genes(Martinez Arias et al, 2002). Thus the correlation between Notch1activation by NB-3 and endogenous Hes1 mRNA level in OLN cells wasinvestigated. Real-time PCR showed that compared to BSA, NB-3 inductionresulted in the similar level of Hes1 mRNA and oscillatory expressionpattern (Ref) (FIG. 15Ca). 48 hours after various treatments, includinganother cell adhesion molecule L1, Hes1 mRNA still remained at basallevel (FIG. 15Cb). To further clarify whether NB-3 activates Hes1, Hesluciferase reporter assay (FIG. 15Cc) was used. Neither Hes1 nor Hes5was activated by NB-3 treatment, while constitutive-active Notch1 (caN1)(Small et al, 2001) activated both luciferase reporters, indicating thatNB-3 did not activate Hes1 and Hes5.

NB-3/Notch Interaction Upregulates MAG Via DTX1

The effect of NB-3/Notch1 signalling in myelination was furtherexplored. The membrane extracts from co-cultured OLN cells with NB-3- ormock-transfected CHO cells were immunoblotted for MAG, a hallmark of OLmaturation. MAG was upregulated only in OLN cells cultured withNB-3-transfected CHO cells (FIG. 16Aa). Real-time PCR showed that NB-3increased MAG transcripts about 24 folds in primary OLs from P5-P7 ratcerebral cortex (FIG. 16Ab). Immunostaining showed that in response toNB-3, mNotch1-myc transfected OLN cells produced a 2.5-fold increase inMAG staining (FIG. 16Ac, f), compared with Jagged1 (FIG. 16Ad-f) or BSA(FIG. 16Af). MAG upregulation was blocked by pre-treating the cells withNotch1 EGF antibody (FIG. 16Af). To study the role of NB-3/Notch1 in MAGupregulation, OLN cells were transfected with dominant-negative Notch1(dn-N1) (Small et al, 2001) that lacks the intracellular portion butstill can bind to extracellular ligands. After treatment with NB-3, MAGupregulation was not observed in transfected cells (FIG. 16Ba, green).Moreover, V1744K or V1744L-transfected OLN cells (green) also abolishedMAG elevation (FIG. 16Bb, c). Since caN1 alone failed to increase MAGexpression in transfected cells (FIG. 16Bd, e, green), theseobservations indicate that NB-3-generated NICD is essential for MAGupregulation.

NB-3-Induced MAG Upregulation Involves DTX1

To identify the transcription factor involved in this event, OLN cellswere transfected with dominant-negative RBP-J (dn-RBP-J) (Kato et al,1997), which lack effective high affinity binding to DNA (FIG. 16Bh) orDTX1 (Yamamoto et al, 2001) (FIG. 16Bi) or two DTXL deletion mutantsthat lack the Ring-H2 motif required for oligomerization of DTX1 (FIG.16Bf, j, k): DTX1-D1 (Yamamoto et al, 2001) and DTX1-D2 (Izon et al,2002). Hes1 luciferase reporter assay confirmed the validity of theseconstructs in OLN cells in that dn-RBP-J inhibited Hes1 transactivationby caN1 and the competitive binding of DTX1 to caN1 also affected this,which was restored by DTX1-D1 or DTX1-D2 (FIG. 16Bg). After NB-3treatment, cells were immunostained for MAG and fluorescence intensitycounted (FIG. 16Bl). It was found that the ablation of Hes1 basalexpression in OLN cells (green) by dn-RBP-J (FIG. 16Bh) did not affectNB-3-induced MAG upregulation. Similarly, DTX1-transfected cells (FIG.16Bi, green) responded to NB-3 stimulation. However, the dysfunction ofendogenous DTX1 by DTX1-D1 and DTX1-D2 (FIG. 16Bj, k, green) abolishedthis event, indicating that DTX1 is involved in NB-3-mediated MAGexpression. Thus NB-3 may activate the Notch1 receptor and release NICD,which recruits DTX1 during myelination.

NB-3 Developmentally Clustering at Paranodes Promotes OPCDifferentiation via Notch1/DTX1 Signaling Pathway

Since NB-3 is expressed from E17, it is conceivable that the two pairsof interaction. Jagged1/Notch1 and NB-3/Notch1 may coexist along theaxon at early developmental stage. To explore whether NB-3 and Jagged1coordinate myelination in a step-wise fashion, the spatial correlationbetween these two molecules along axons during development was firststudied. Sections of P2, P5, and P14 (not shown) rat brain stems weredouble labelled for Jagged1 (green) and NB-3 (red). At P2, Jagged1 wasevenly distributed along the axon and congregated NB-3 was hardlydetectable (FIG. 17Aa), which was in agreement with the previousobservation that Jagged1/Notch1 signalling may predominate to promotemigration of young OL loops along axons at this stage (Wang et al,1998). At P5, NB-3 clustered at the paranode (FIG. 17Ab, c) and Jagged1occupied the juxtaparanode and internode, separated from paranodal NB-3(FIG. 17A). Given the decrease of Jagged1 expression (Wang et al, 1998)after P6, the inventor's observations suggest that the balance betweenthese two pairs of interactions may be disrupted during the later stageof OL maturation, in other words, NB-3/Notch1 interaction predominatesto induce OL maturation at the CNS paranode. To attest this notion,purified OPCs from P7 Wistar rat optic nerve (FIG. 17Ba) were treatedwith BSA, NB-3 or Jagged1 for 2 days and then immunostained for Ng2(red), a progenitor marker and CNPase (green), a young OL marker (FIG.17Bb-d). Statistical counting showed that NB-3 promoted OPCdifferentiation into CNPase-positive OLs (˜75%), compared to BSA (˜50%)or Jagged1 (˜0%) treatment (FIG. 17Bh). To further confirm theinvolvement of Notch1 and DTX1 in NB-3-promoted OPC differentiation,OPCs were transfected with dn-N1 (FIG. 17Be) or DTX1-D1 (FIG. 17Bf) andtreated simultaneously with NB-3. Double labelling for tags (green) andCNPase (red) showed that introduction of either construct significantlyblocked NB-3-promoted OPC differentiation into OLs (FIG. 17Bh). Andconsistent with the previous study (Wang et al, 1998), caN1-transfectedOPCs remained undifferentiated (FIG. 17Bg, h).

NB-3/Notch Signalling Via Deltex1 Directs Differentiation of EmbryonicNeural Stem Cells Into Oligodendrocytes

To explore whether NB-3/Notch1 interaction is also involved inoligodendrogliogenesis from Neural Stem Cells (NSCs), the developmentalexpression patterns of NB-3 and Notch1 was investigated. Western blot ofrat brain samples showed that both NB-3 and Notch1 were expressed atembryonic day 17 (E17) and an abrupt increase after birth reached amaximum level between postnatal day 0 (P0) and 21 (P21), whichcorresponds to the time frame of oligodendrogliogenesis from NSCs (FIG.18).

NSCs: NB-3 is a Functional Ligand of Notch1

The expression of Notch1 on NSCs by immunofluorescence was studied next.The NSCs used in this study were isolated from embryonic day 14 BALB/cmouse embryo striatum (Arsenijevic et al, 2001). These cells expressedintermediate filament protein nestin (FIG. 19Aa), a progenitor marker,and Notch1 (FIG. 19Ab, c). To confirm the NB-3/Notch interactionpreviously observed, NB-3-Fc fusion protein coupled to Protein A beadswas used to precipitate binding partner of NB-3 from NSC membraneextracts. The precipitate was positively blotted with Notch1 antibody(FIG. 19Ba). Further, P0 rat brain membrane extracts wereimmunoprecipitated using NB-3 antibody and blotted with Notch1 antibodyand vice versa (FIG. 19Ba). Western blot showed that NB-3 and Notch1co-immunoprecipitated. Moreover, cell adhesion assay was performed byplating murine Notch1—(N1) and mock-transfected HeLa cells onto NB-3substrate coated proteins. N1-transfected HeLa cells adhered to NB-3(FIG. 19Bb), but mock-transfected HeLa cells did not bind (FIG. 19Bf).Adhesion was prevented by pre-incubation of cells with NB-3 or Notch1antibodies (FIG. 19Bc, d), but not with pre-immune serum (FIG. 19Be).These studies corroborate that NB-3 is a binding partner of Notch.

NSCs: NB-3 Induces NICD Nuclear Translocation but Does Not Activate Hes1

Notch activation by ligand binding is featured by NICD nucleartranslocation (Schroeter et al, 1998). The inventor studied whether NICDnuclear translocation in NSCs could occur in response to NB-3stimulation. It has been observed above that NB-3 induces γ-secretasedependent NICD nuclear translocation in OLN cells. In agreement withthat study, merged triple staining showed that NB-3 treatment of nestin(green) positive NSCs resulted in NICD clustering (red) in the nucleusvisualized by Hoechst 33258 staining (blue) (FIG. 19Ca), which wassimilar to Jagged1 stimulation (FIG. 19Cb). However, BSA failed totrigger this event (FIG. 19 Cc), suggesting that NB-3 specificallyinteracts with Notch1 to effect typical NICD nuclear translocation.Another prominent feature of classic Notch signalling pathway is thatnuclear NICD transactivates target genes, such as Hes genes (MartinezArias et al, 2002). Thus the Hes1 luciferase reporter assay was utilizedto investigate whether NB-3-generated NICD could activate Hes1. Theresults showed that NB-3 did not upregulate Hes1, while either Jagged1or constitutive-active Notch1 (caN1) (Small et al, 2001) activated Hes1as expected (FIG. 19Da). As increasing evidence showed that Deltex1(DTX1) is another Notch downstream element (Yamamoto et al, 2001), theimpact of NB-3 on DTX1-mediated Mash1 transcriptional activity in pE7luciferase reporter assays was studied (FIG. 19Db). Consistent with theprevious report (Yamamoto et al, 2001), DTX1 inhibited Mash1transactivation of pE7 reporter. However, NB-3 treatment mimicked thisinhibition partly, which was abolished by one DTX1 deletion mutant,Flagged-tagged DTX1-D2 (Izon et al, 2002) that lacks the domain 3,Ring-H2 motif which is required for functional DTX1 homodimer formation(Matsuno et al, 2002) (FIG. 21 c). These results suggest thatNB-3-generated NICD may mediate DTX1-related transcription events.

NSCs: NB-3 Promotes OL Generation

Given that the expression of both NB-3 and Notch1 parallel the timeframe of OL development, the inventor explored whether NB-3/Notchinteraction was involved in oligodendrogliogenesis from NSCs. To dothis, NSCs were allowed to differentiate for 7 days in the absence ofmitogen and in the presence of serum and NB-3 (FIG. 20 a, d), BSA (FIG.20 b, e), or Jagged1 (FIG. 20 c, f). The cells, identified by Hoechst33258 (FIG. 20 a-f, blue), were immunostained for OL marker: CNPase(FIG. 20 a-c, red); neuronal marker: β-tubulin (FIG. 20 d-f, red); andastrocyte marker: GFAP (FIG. 20 a-f, green). The results showed thatdistinct from BSA and Jagged1, NB-3 promoted OL generation. On the otherhand, compared to BSA, NB-3 had little impact on neurogenesis, whileJagged1 inhibited this as reported (Morrison et al, 2000). Flowcytometry confirmed these observations in that NB-3 induced a 2-foldincrease in OL generated, compared to BSA treatment, while Jagged1inhibited OL development (FIG. 20 g).

NSCs: NB-3/Notch Signalling Pathway Via DTX1 InstructsOligodendrogliogenesis

To confirm the involvement of Notch1 in this event, NSCs weretransfected with V5-tagged dominant-negative Notch1 (dn-N1) (Small etal, 2001). dn-N1 lacks the intracellular domain but can still bind tothe extracellular ligand. The Hes1 luciferase reporter assay showed thatdn-N1 failed to respond to Jagged1 treatment to activate Hes1 (FIG. 21h). Mitogens were then withdrawn and NB-3 added to the culture medium.Double labeling for V5 and CNPase or GFAP showed that dysfunction ofNotch1 by dn-N1 abolished NB-3-promoted oligodendrogliogenesis, whilefavouring astrocyte formation (FIG. 21 a, b, i). To further investigatewhether DTX1 participates in NB-3-induced OL formation, NSCs weretransfected with Flag-tagged DTX1-D2 (FIG. 21 c). Hes1 luciferasereporter assay confirmed that DTX1 inhibited Hes1 activation by caN1while DTX1-D2 restored Hes1 elevation by caN1 (FIG. 21 h). Afterdifferentiation in the presence of NB-3, NSCs were immunostained forFlag and CNPase or GFAP. The results showed that DTX1-D2 transfectedcells failed to differentiate into OL after NB-3 stimulation but weredirected to astrocytes (FIG. 21 d, e, i). And consistent with previousstudies (Tanigaki et al, 2001) introduction of caN1 into NSCs resultedin astrogliogenesis while inhibiting OL generation (FIG. 21 f, g, i).These observations indicate that NB-3 promoted oligodendrogliogenesisvia Notch/DTX1 signalling pathway (FIG. 22).

Discussion Relating to the Second Aspect of the Invention

Using molecular, cellular, and morphological approaches, the inventorhas identified a functional molecular interaction between Notch andF3/NB-3 at the paranode, a vital site for axoglial signalling inmyelination. A new axonal adhesion molecule—NB-3, has been localized tothe paranode. This interaction may regulate the differentiation of themyelinating oligodendrocyte during axonal ensheathment and act toprevent putative nodes of Ranvier from becoming invested in myelin. Inaddition to established Notch/Jagged1 signaling pathway, the inventorprovides evidence for novel Notch ligands—F3 and NB-3 in the context ofthe myelinating process, suggesting that signaling via Jagged1 andF3/NB-3 may contribute in a co-ordinated fashion to myelination.

The data designates the adhesion molecule F3 as a new functional ligandof Notch. F3/Notch interaction induces the generation and nucleartranslocation of NICD and elevates Notch1 and Notch2 expression. Itpromotes OPC differentiation and upregulates MAG in OLN cells andprimary OLs, thus revealing a potential regulatory role for F3 in OLmaturation. Thus, ligand-specific Notch signaling via Jagged1 as aninhibitory factor and F3 as a positive instructor may regulatemyelination in a coordinated fashion (FIG. 13).

The data also designates NB-3 as another new functional ligand of Notch.The extracellular NB-3/Notch interaction releases from the membraneNICD, which recruits DTX1 and translocates into the nucleus where thecomplex mediates directly or indirectly CNPase expression, thuspromoting oligodendrogliogenesis (FIG. 22).

Notch is an Oligodendroglial Surface Receptor of F3/NB-3

Intuitively, both axons and oligodendrocytes must actively participateand achieve two-way communication if myelination is to be properlyco-ordinated. The emerging concept is that of an active signalingaxoglial channel sited at paranodes, where cytoplasm-filled glial loopsspiral and indent the adjacent axolemmal membrane. Although paranodalaxonal and glial molecular members have been described, their functionalinteractions in regulating myelination remain ill-defined. In support ofthe notion that glial Notch could act as the receptor for axonal F3 andNB-3, the inventor shows through co-immunoprecipitation experiments andGST pull-down assays that these molecules physically interact.Furthermore, he has identified two regions of F3 interaction (N1.1 andN1.3) in the extracellular domain of Notch1, one of which (N1.3) isshared with or overlaps with that involved in NB-3 binding.

Notch is a Functional Receptor for F3/NB-3

This study proposes the presence of a molecular stop signal to preventmature myelinating oligodendrocytes from encroaching upon andmyelinating putative nodes of Ranvier. The inventor has direct evidenceof oligodendroglial processes emanating from OLN-93 cells terminatingand spreading over the surface of F3 transfected CHO cells. Themolecular basis for this signal is an interaction between F3 and Notch.In support of this, adhesion assays using OLN-93 and Notch1-transfectedHeLa cells cultured upon a F3 substrate demonstrate that cellularinteractions are disrupted by specific antibodies against the proposedligand-receptor pair. Together with previous morphological data thatconfirm the presence of F3 at the paranodes and the inventor's data thatreveal surface expression of Notch receptors on oligodendrocytes, theinventor suggests that an F3 signalling interaction indeed exists at theparanode and acts to prevent further extension of oligodendroglialprocesses. This interesting outcome is unique in postulating a mechanismthat regulates the lateral limits of the myelinated internodes. In asimilar manner to F3, the inventor has also provided evidence that NB-3is a ligand for Notch1. Plating the Notch1-expressing HeLa cells on F3or NB-3 led to different interactive outcomes; adherence to the NB-3substrate, but repulsion from the F3 substrate. This could be due todistinct signals originating from the dual site F3-Notch interaction(resulting in cell repulsion) and the single site NB-3-Notch interaction(mediating adhesion). OLN-93 cells plated on NB-3 undergo a rapiddifferentiation and morphological alteration which ultimately leads to aflattened sheet-like appearance of the cell body, possibly representinga differentiation step in the multi-phase process of myelination as theoligodendrocytes prepare to ensheath contacting axons. However, thecommon theme underlying the F3/Notch and NB-3/Notch interactions is thatboth are linked to myelination as evidenced by the upregulation of MAGat both protein and mRNA levels.

Different Ligands Signal to Glial-Derived Notch to Co-ordinateMyelination

The complex nature of myelination demands a co-ordinated, dynamic seriesof axon-glial interactions which are likely regulated by the temporaland spatial distribution of trans interacting components. One suchregulated interaction involves the interaction of axonal Jagged1 andoligodendrocyte-derived Notch1 in rat optic nerve (Wang et al, 1998).Notch receptor activation by Jagged1 inhibits differentiation andpromotes migration of oligodendrocyte cellular processes along the axon.In an optic nerve model, Jagged1 is downregulated with a time coursethat parallels myelination. In accord with the finding that myelinationbegins only after target innervation (Schwab and Schnell, 1989), furtherstudies showed that Jagged1 downregulation occurred only after axonsreached their targets (Dugas et al, 2001). The Jagged1/ Notch signalthat keeps oligodendrocytes in an immature state thus abates anddifferentiation of oligodendrocytes into mature myelin-forming cellsprogressively occurs. This key signal may then allow contact andextension of oligodendroglial cellular processes along the axon. Theinventor's results support the idea that the Notch receptors may switchto other ligands, such as clustered F3/NB-3 as positive signals atparanodes, triggering the onset of the ensheathing process. This ligandswitch mechanism may also underlie a phase transition fromdifferentiating oligodendrocytes held in check by an axonal inhibitorysignal to subsequent maturation into myelinating cells.

Spatial regulation of myelination may be achieved by the clustering ofmolecules along the axon. Clustering of sodium channels has beendescribed (Salzer, 1997). In the maturing nerve, Caspr molecules areprogressively herded towards the node to form tight bands, the “Casprspiral”, on either side of it (Pedraza et al, 2001).

Oligodendrocytes themselves appear crucial in promoting paranodeformation through directing molecular localization. FIAU-inducedablation of oligodendrocytes in MBP-TK led to mis-localization of Caspr(Mathis et al, 2001). Significantly, the few myelinatingoligodendrocytes in the MBP-TK brains were associated with focalclusters of Caspr close to MBP-positive domains. In jimpy mice,spontaneous degeneration of oligodendrocytes and mis-localization ofCaspr occurs (Mathis et al, 2001). The inventor's previous work has alsoshown in re-myelinating peripheral nerves how F3, initially diffuselydistributed along the demyelinated nerve fibre, becomes clustered at theparanodes by the effect of re-myelinating Schwann cells. Specifically,F3 clustering is seen at the extending edge of the Schwann cell(Kazarinova-Noyes et al, 2001). The results provided herein suggest thatNB-3 also undergoes a similar clustering process, as it is barelydetectable along P2 axons but forms distinct pairs of bandscorresponding to paranodal staining at P5 and P14 (FIG. 4). This resultalso supports the notion that oligodendrocytes may be required forclustering of axonal molecules, such as Caspr, F3, and NB-3.

The oligodendroglial-dependent clustering may not only result in aredistribution of axonal proteins into domains but also lead to a “doseeffect”, in that molecules that were once sparsely distributed becomepacked together in highly dense bands. Only the concentrated axonalligand may productively signal to glial receptors to achieve the “stop”effect, explaining why stop signals are not prematurely activated at theinitial phase of axoglial contact when the axonal molecules arediffusely distributed. In this way, clustering could promote the switchof axonal cues, for example for Notch receptor switching from internodalJagged1 to paranodal F3 and NB-3. This switch in cues would represent anessential regulatory mechanism during axonal ensheathment. In essence,the myelinating oligodendrocyte can be likened, albeit simplistically,to a machine with excavators at its lateral tips, its motor enginestarted (oligodendrocyte differentiation triggered by downregulation ofJagged1/Notch inhibitory signal), heaping up larger and larger mounds ofearth as it progresses forwards (clustering of nodal and paranodalmolecules) and then braking to a halt via an in-built feedback mechanism(clustered “high dose” F3 signalling to oligodendroglial Notch).

A number of studies have addressed signalling mechanisms that may play apart during myelination, such as the roles of neurofascin(Martin-Collinson et al, 1998) and N-cadherin (Schnadelbach et al,2001). The inventor favours the concept proposed by Pedraza et al (2001)that membrane proteins of the axoglial junction at paranodes, as aconduit for bi-directional signal transduction, may act as selectivemolecular sieves and diffusion barriers whose purpose is to contributeto the organization of axonal domain architecture. Membrane proteins ofthe apposing axolemma and glial membrane loop interact via adhesionmolecules to form mobile constructs of molecular sieves and barriersthat “travel” along the axolemmal surface, pushing sodium channels andpacking them at the nodes and allowing potassium channels to passthrough to reach the juxtaparanode. Intracellular proteins linked to thecytoplasmic domains of the axolemmal and glial molecules may alsocontribute in a significant fashion to this barrier/sieving effect.Given that both F3 and NB-3 are GPI-linked molecules on the axonalsurface, it will be interesting to investigate their cis interactionswith transmembrane proteins and the nature of associated intracellularsignal transduction pathways. Caspr, which associates with F3, interactswith members of the protein 4.1 family (Baumgartner et al, 1996; Ward etal, 1998) via its intracellular segment. This connects Caspr to spectrinand the axonal cytoskeleton (Hoover and Bryant, 2000). The presence ofintact nodes of Ranvier containing clustered sodium channels butabnormally distributed F3, Caspr and potassium channels in theCGT-deficient mouse testifies to the fact that other importantsignalling mechanisms may exist (Coetzee et al, 1996; Bosio et al,1998). As this enzyme (UDP-galactose-ceramide galactosyltransferase) isrequired for galactolipid synthesis, lipid molecules may also be crucialregulators of myelination. Further work to define the configuration andcomponents of the axoglial junction will no doubt help create aknowledge base from which it is hoped that meaningful strategies topromote re-myelination of the damaged CNS will arise.

This study also reveals a new facet of Notch signalling—the existence ofF3 and NB-3 as signalling ligands. These neural cell adhesion moleculesare relatively widely distributed in the nervous system and may regulateNotch mediated processes. At present, Notch receptors are mostlyinvestigated in the context of DSL (Delta, Serrate, Lag-2) ligands, CSL(CBF1, Suppresssor of hairless, Lag-1) transcriptional cofactors andgene targets which are categorized as the HES (Hairy/ Enhancer of Split)family of basic helix-loop-helix transcriptional regulators. The Notchreceptor classically plays a critical role in cell fate selection duringdevelopment (Artavanis-Tsakanos et al, 1999; Baker, 2000; Munm andKopan, 2000). Apart from neurogenesis during development, Notch alsoparticipates in gliogenesis (Morrison et al, 2000) and has beenimplicated in T-lymphocyte development (Robey et al, 1996),haematological malignancies (Ellisen et al, 1991) and familial strokesyndromes (Joutel et al, 1997). Notch is also unique as one of a fewproteins that undergo regulated intramembrane proteolysis (Weinmaster,2000). The modulation of these processes by cell adhesion molecules thusbecomes another avenue for research.

Notch is an Oligodendroglial Surface Receptor of F3

Adhesive contacts at axoglial junctions are partly contributed by theF3-Caspr-neurofascin 155 heterotrimer (Girault and Peles, 2002), but theexact role of this complex remains to be further characterized. Hereinthe inventor defines a novel ligand-receptor interaction between F3 andNotch. Cell adhesion/repulsion assays and biochemical approachesdemonstrate that Notch and F3 are binding partners. Further twoextracellular sites on Notch1 for F3 binding have been identified,namely, N1.1 and N1.3. The former contains the EGF repeats 11-12involved in DSL binding (Rebay et al, 1991). F3 is also expressed on OLs(Koch et al, 1997) and transduces signals to glial intracellular Fynwhich then interacts with Tau protein to mediate myelination (Klein etal, 2002). Since soluble F3 is sufficient to trigger F3/Notch signalingand the lipid raft assay demonstrated that Notch1 is not localized tothe F3-enriched fraction, F3 may not interact with Notch1 in cis. Giventhat Notch and F3 co-localize in various regions of the brain (Lardelliet al, 1994; Revest et al, 1999), particularly at axoglial junctions,the results suggest a role for F3 as a trans-acting ligand of Notch.

F3/Notch Signaling Induces Proteolytic Release of NICD and UpregulatesNotch Homologs

RIP, the generation of nuclear signaling proteins derived fromnon-nuclear precursors such as Notch and APP, is a new paradigm ofsignal transduction that potentially adds unforeseen diversity to thesignaling repertoire of a cell (Ebinu and Yankner, 2002). As with DSL,the inventor has shown that F3 binding triggers Notch intramembraneproteolysis at the S3 site and nuclear translocation of the resultantNICD in OLN cells. The ability of Notch1 EGF (EGF 11-12) antibody, orbrefeldin A and monensin, to block this event suggests that theextracellular F3/Notch interaction is essential for the intramembranecleavage-derived generation and transport of NICD. Thus, it is anotherexample of RIP. In addition, the F3/Notch signaling pathway may activatea feedback loop that specifically increases Notch1 and Notch2 (but notNotch3) expression or protects Notch1 and Notch2 from rapid degradation.Either way, this may serve to replenish consumed receptors on the cellsurface and thus sustain signal continuity.

A Possible Model for F3/Notch Signaling Via DTX1

Given that F3 and Jagged1 share a common binding domain on Notch1,Jagged1 downregulation that occurs prior to myelination may act topermit the alternate interaction of Notch with F3. In OLN cells, bothJagged1 and F3 trigger γ-secretase-mediated and S3-directed release ofNICD followed by its nuclear translocation. Moreover, caN1transactivates Hes1, which is blocked by co-expression of dn-RBP-J-mycor DTX1. Thus it is conceivable that F3-induced Notch intracellularsignaling is associated with RBP-J. However, only F3-induced NICD, butnot Jagged1-induced NICD or caN1 and caN2, increases MAG expression,suggesting that this event may require specific extracellularligand-receptor interaction. Moreover, experiments utilizing eitherdn-RBP-J-myc or Hes1 antisense oligonucleotides indicate that theblockage of endogenous Hes1 expression is not involved inF3/Notch-induced MAG upregulation. On the other hand, two truncatedmutants of DTX1, which lack the Ring-H2 finger motif that isindispensable for the formation of functional homodimeric DTX1 (Matsunoet al, 2002), prevent both F3-promoted OPC differentiation and MAGupregulation. Thus it is proposed that a switch in ligands may alterNotch intracellular signaling effectors (FIG. 13). The binding ofdifferent ligands may induce the formation of distinct Notchconformations. Such conformational alterations could result fromdifferent, albeit overlapping, regions of Notch recognized by Jagged1and F3. Notch receptors with distinct conformations may interact, beforeor after cleavage, with different cytoplasmic factors, such as DTX1. Theform of NICD subsequently arriving at the nucleus then specifies furtherpotential interactions and determines its transcriptional target. Itwill be of significance to investigate this hypothesis and identifyDTX1-related transcription cofactors that are required for F3/Notchsignaling.

Potential Role of F3/Notch Signaling Via DTX1 in OL Maturation

Jagged1/Notch signaling contributes to maintaining OPCs in anundifferentiated stage (Wang et al, 1998). The failure of efficientremyelination in multiple sclerosis (MS) has been partly attributed tothe activation of OPC Notch by astrocyte-expressed Jagged1 (John et al,2002). However, Jagged1 expression sharply decreases from around P6(Wang et al, 1998), a time point concurrent with the onset ofmyelination and the clustering of axonal F3 at the paranode(Kazarinova-Noyes et al, 2001), an ideal position to interact with Notchreceptors on the surface of newly formed OLs. Mutant animals, in whichNotch1 is selectively ablated in OPCs (Genoud et al, 2002), arecharacterized by ectopic immature OLs, most of which undergo apoptosis,indicating that autonomous differentiation in the absence of the Notchreceptor may be disruptive and that other regulatory signaling cascadesbesides Jagged1/Notch may be needed to ensure correct differentiationand survival of OLs. It was observed here that F3 promotes OPCdifferentiation, which can be blocked by both dominant-negative Notchand DTX1 deletion mutants, and oligodendroglial processes emanating fromOLN cells terminate and spread over the surface of F3-transfected CHOcells, an event related to the upregulation of myelin-specific proteins,such as MAG and CNPase. Upon Notch interaction with either immobilizedor soluble F3, MAG, a specific marker of mature OLs, is significantlyupregulated at both the protein and mRNA levels. MAG upregulation can beblocked by dominant-negative construct of Notch1, Notch2, or deletionmutants of DTX1. These observations indicate that OL maturation involvesF3/Notch signaling via DTX1 (FIG. 13).

In summary, the study reveals a new facet of the Notch signalingpathway. Upon activation by F3, Notch signaling via DTX1 continues toparticipate in OL maturation through upregulating certain myelin-relatedproteins instead of solely functioning to inhibit OPC differentiationinto OLs. Hence, this finding may prove to be an efficient molecularhandle for promoting remyelination in degenerative diseases, such as MS,by creating an environment in which Notch predominantly interacts withendogenous or exogenous F3 to initiate the F3/Notch/DTX1 signalingpathway.

NB-3 is a New Constituent of the Paranodal Architecture

The construction of a myelinated axon during nervous system developmentis a well-orchestrated phenomenon, yet the molecular details of thisprocess remain unclear. Apart from the segmental nature of myelinensheathment, a redistribution of axonal molecules occurs to createdistinct axonal domains in the mature nerve fibre. The moleculararchitecture of the various axonal domains has been extensively reviewed(Morell and Quarles, 1999; Arroyo et al, 2000; Peles and Salzer, 2000;Denisenko-Nehrbass et al, 2002). Constituents of the paranode includeaxonal F3 in association with the neurexin superfamily molecule, Caspr,and oligodendroglial neurofascin-155 (NF-155) (R10s et al, 2000; Tait etal, 2000). Studies of F3-null mice highlight F3 as a key player in thesynthesis of the axoglial apparatus in the peripheral nervous system(PNS). Prior to their death by postnatal day 18, the mutant mice have anataxic phenotype with hindlimb weakness, attributed to defectiveconnections involving cerebellar interneurons and to reduced nerveconduction velocity and excitability (Berglund et al, 1999; Boyle et al,2001). The latter problem with nerve impulse conduction reflectsabnormalities in axoglial junction formation in myelinated peripheralnerve. Consistent with a paranodal function of F3, these abnormalitiesinclude disruption of the axoglial junction and defective transport ofCaspr, a cis binding partner of F3, to the axon membrane (Boyle et al,2001). The fact that adhesive axoglial interactions still form in the F3mutant mice points to the involvement of other paranodal molecules, withNB-3 being one such candidate. Although the distribution/localization ofNB-3 in the PNS is unknown, in the CNS NB-3 is expressed in theolfactory bulb, layers I, III and V of the cerebral cortex, piriformcortex, anterior thalamic nuclei, locus coeruleus, mesencephalictrigeminal nucleus and the Purkinje cells of the cerebellum (Lee et al,2000). The inventor shows that NB-3 clusters and co-localizes with Casprat paranodes from P5 to adulthood, certainly suggesting the likehood ofit being similarly localized in the PNS.

NB-3 Clustering at the CNS Paranode Promotes Maturation ofOligodendrocyte Precursor Cells Via Notch/Deltex1 Signalling Pathway

In summary, the above study has shown that NB-3, a neuron-derivedmolecule, is a functional ligand of Notch1. Acting as a spatial switchsignal by developmentally clustering at the CNS paranode, it releasesNICD at the S3 site via RIP and triggers Notch/DTX1 signalling pathway,which promotes OPC differentiation and OL maturation to coordinatemyelination.

NB-3/Notch Signalling Via Deltex1 Directs Differentition of EmbryonicNeural Stem Cells Into Oligodendrocytes

Multiple sclerosis (MS) is a chronic demyelinating disease in the CNS.Neural stem cell transplantation is a promising treatment for suchdiseases (Pluchino et al, 2003). It will be of significance to establisha reliable method for selectively predifferentiating appropriate donorstem cells to OPCs or OLs. In the present study, pre-clinical validationhas been provided of a novel signalling pathway allowing for the firsttime using NB-3, a neuronal cell recognition molecule, to directneuronal stem cells to OPC and/or OLs lineages in vitro. Moreover,recent studies have shown that the failure of efficient remyelination inMS is partly attributed to the activation of OPC Notch receptor byastrocyte-expressed Jagged1 (John et al, 2002). The ectopic immature OLsand subsequent apoptosis appear in mutant mice in which Notch1signalling is selectively inhibited in OPCs (Genoud et al, 2002). Theseobservations implicate that, in addition to Jagged1/Notch1 interaction,there may exist other axon-derived Notch1 ligands that mediate OLdifferentiation by properly activating the Notch1 signalling pathway (Huet al, 2003). In support of this notion, the inventor's presentobservations in NSCs incriminate that NB-3/Notch signalling via Deltex1may modulate OL differentiation from NSCs and thus represent a potentialtarget for therapeutic intervention in demyelinating diseases.

Materials and Methods Relating to the Second Aspect of the Invention

Antibodies

Monoclonal Notch1 EGF repeats 11-12 (Neomarker), CNPase, Flag,beta-tubulin and MAP2 (2a+2b) (Sigma), c-myc (9E10) and HA (Santa CruzBiotechnology), nuclear matrix protein, Gal-C and nestin (Chemicon),GFAP (DAKO) and V5 epitope (Invitrogen) antibodies; polyclonalV1744-cleaved NICD (Cell Signaling), N21CD, Jagged1, Jagged2, Delta(Santa Cruz Biotechnology), γ-tubulin (Sigma), Ng2, sodium channels(NaCh), NF200 (Chemicon), Hes1 (Kaneta et al, 2000), NICD (Logeat et al,1998), Notch1, Notch2, Notch3 (kind gifts from Dr. Lendahl) and F3(Shimazaki et al, 1998).

Polyclonal Caspr antibody was obtained by immunization of rabbits with aGST fusion protein of amino acids 277-430 of human Caspr and polyclonalMAG antibody (7610) by immunization of rabbits with the followingpeptide: N′-CISCGAPDKYESREVST-C′ (Eurogentec).

Secondary antibodies conjugated to Cy2, Cy3 and FITC were obtained fromAmersham Pharmacia Biotech. Inc.

Anti-NB-3 serum and monoclonal antibodies were generated againstrecombinant protein expressed in E. coli transformed with the pET15bvector (Novagen) containing rat NB-3 cDNA encoding Ig domains I-II(amino acids 30-227). To construct this expression vector, twooligonucleotide primers were used in PCR reactions to amplify cDNAencoding the corresponding region from a cDNA library synthesized fromrat brain total RNA. The two oligonucleotide primers are:

5′-TCCGGATCCCATGGAGCCACAGGATGTCATTTT-3′ (forward)

5′-TCCGGATCCGTCGACTGGCACATATTCCCCCATGA-3′ (reverse),

The PCR was carried out for 30 cycles at 94° C. for 30s, 60° C. for 30sand 72° C. for 45s after denaturation at 94° C. for 3 min. The amplifiedcDNA fragments were digested with BamHI and inserted into a BamHI-cutpET15b vector after DNA sequencing. The protein was expressed in E. coliBL21(DE3) pLysS by induction with IPTG. It was partially purified asinclusion bodies, and these were solubilized and applied to SDS-PAGE andthe recombinant protein eluted electrophoretically from the gel. Theprotein was used to immunize rabbits for antiserum and BALB/c mice formonoclonal antibodies (Harlow, 1998).

Cell Co-Culture

F3 transfected CHO cells (Gennarini et al, 1991), TAX, TAG-1 and mocktransfected CHO cells (Furley et al, 1990; Tsiotra et al, 1993) wereco-cultured with the oligodendrocyte cell line OLN-93 (Richter-Landsbergand Heinrich, 1996) in a 2:1 ratio in Dulbecco's modied Eagle's medium(DMEM, Gibco), 10% fetal calf serum (Gibco) and penicillin/streptomycin(Gibco) for 2 days at 37° C. in a humidified atmosphere. Cells werepre-stained with the PKH26 red fluorescent cell linker kit (Sigma). Thecells were also stained with primary monoclonal antibodies to c-myc(Santa Cruz Biotechnology) and Cy-2 labelled goat anti-mouse secondaryantibodies (Sigma). To determine the number of the stopped processes inthe co-cultures, OLN-93 cells (more than 100 cells) from three randomlyselected areas in each of three cover slips were counted per experiment.Raw data from at least three independent experiments were analyzed byanalysis of variance and then Newman-Keuls test with p<0.05 and p<0.01being considered significant or highly significant, respectively.

NB-3- or mock-transfected CHO cells were cultured with OLN in a 2:1ratio for 2 days. The membrane portions of the co-cultures wereextracted as described (Xiao et al, 1996).

Transfection and Characterization of NB-3-Transfected CHO Cells

CHO cells were transfected with 10 μg of pcDNA3-NB-3 using lipofectin(GIBCO BRL) according to the manufacturer's instructions. Following drugselection with G418 (Life Technologies, Inc), surviving cell clones wereexpanded and analyzed by Western blot and immunohistochemical stainingfor surface NB-3 expression.

Transfection of Cells

OLN cells were transiently (V1744K-myc, V1744L-myc, dn-N-1-V5, dn-N2-V5,pcDNA4/V5/LacZ (Invitrogen), dn-RBP-J-myc, DTX1-myc, DTX1-D1-HA,DTX1-D2-Flag, caN1, caN2) or stably (mNotch1-myc) transfected usingLipofectamine (Invitrogen). CHO cells were stably transfected withfull-length Jagged1 (Small et al, 2001) using Lipofectamine. The stabletransfectants were screened with 400 μg/ml G418 (Sigma) or 250 μg/mlZeocin (Invitrogen) and identified by immunostaining and Western blot.

Immunocytochemistry

Cells were cultured on 13 mm coverslips (Nalge Nunc International).After various treatments, including γ-secretase inhibitor (Sigma), cellswere fixed with 4% paraformaldehyde and blocked with 1% BSA. Cells werethen incubated with primary antibodies in 0.2% BSA for 1 hour, followedby Cy3-labeled or Cy2-labeled secondary antibody (Sigma). After mountingin fluorescent mounting medium (DAKO), cells were visualized with aLeica DM RXA2 fluorescent microscope. The photos were taken using thesame optical parameters to ensure the comparable luminosity. At leastten different viewing fields from three independent experiments wereused to calculate the percentage of cells showing NICD translocation ordifferentiation. Two hundred cells from at least three independentexperiments were quantified for fluorescence intensities by AdobePhotoshop™ (Jack et al, 2001) and measured for cytoplasmic area by LeicaQFluoro software. The raw data were analyzed by Student's t test withp<0.05 and p<0.01 being considered a significant or highly significantdifference, respectively.

For detection of primary NF200, GFAP and Gal-C antibodies, Alexa Fluor488-conjugated anti-mouse IgG or Alexa Fluor 546-conjugated anti-rabbitIgG (1:1500; Molecular Probes) were used. For transfected NSCs, cellswere first transfected with plasmid caN1, DTX-D2, dnN1 usingLipofectamine 2000 (Invitrogen) and treated with or without NB-3 inserum free culture medium without growth factor for 24 hours, and thencells were allowed to differentiate on 13 mm coverslips in 1% FCSmedium.

Cell Adhesion and Repulsion Assay

This was carried out as previously described (Xiao et al, 1996).Briefly, 35 mm tissue culture petri dishes (Becton Dickinson) werecoated with methanol solubilized nitrocellulose (Lagenaur and Lemmon,1987) and air-dried under a sterile hood. Proteins (2.5 μl of 12 μMF3-Fc, CHL1-Fc, or different subcloned Notch1 extracellular fragments)were then applied to these dishes and incubated for 2 hours at 37° C. ina humidified atmosphere. Subsequently, the dishes were washed threetimes with calcium- and magnesium-free Hank's balanced salt solution(CMF-HBSS) and blocked overnight with 2% heat-inactivated fattyacid-free BSA (Sigma) in CMF-HBSS (this blocking step is skipped in therepulsion test). The dishes were then rinsed again and the respectivecells, such as OLN-93, murine Notch1 transfected HeLa cells (Logeat et.al., 1998), F3-transfected CHO cells, and NB-3-transfected CHO cells,were plated in 2 ml of chemically defined medium at a density of 1.5×10⁶cells/ml. After 0.5 hour (in the adhesion test) or 12 hours (in therepulsion test), the dishes were gently washed three times with CMF-HBSSand the cells were fixed with 2.5% glutaldehyde in CMF-HBSS. Blockage ofadhesion was carried out using anti-F3 (Gennarini et al, 1991; 1:100),polyclonal anti-Notch1 (Mitsiadis et al, 1995; 1:200) and anti-Notch2(Mitsiadis et al, 1995; 1:200) antibodies. Cells adhering to the variousspots were photographed and counted. The results were analyzed byStudent's t test with p<0.05 and p<0.01 being considered significant orhighly significant difference, respectively.

Western Blot Analysis

Co-cultures of F3, TAX, TAG-1 and mock transfected CHO cells with OLN-93cells were harvested and lysed by sonication in PBS containing theprotease inhibitor cocktail tablet. After centrifuging at 100,000 g for1 hour at 4° C., the pellets were further solubilized in 2% TritonX-100. Subsequently, the membrane portion of the cells, about 20 μgprotein per cell line, was analyzed by SDS-PAGE (8% gels; Laemmli, 1970)and Western blotting (Towbin et al, 1979) with antibodies againstNotch1, Notch2, Notch3, myelin-associated glycoprotein, MAG (Yang et al,1999) and proteolipid protein (PLP) (Jung et al, 1996).

Western Blot Analysis of Developmental Expression Patterns

The brain stem was dissected from embryos at E17 or neonates at betweenP0 and P21 of Wistar rats. The specimens were homogenized in 9 volumesof reducing sample buffer and boiled for 5 min. Each 10 μl aliquot ofthe homogenates was subjected to Western blot. Detection was carried outwith ECL Western Blotting System (Amersham Biosciences).

Fusion Proteins

Production of recombinant F3-Fc, Jagged1-Fc and CHL1-Fc fusion proteins.Recombinant cDNA encoding mouse F3 with the GPI-anchor substituted withhuman IgG Fc was inserted at the Hind III-Not I sites of pDX with anamplification-promoting sequence (APS) (Hemann et al, 1994) andintroduced into Ltk−/− cells. Fc fusion proteins were purified asdescribed (Shimizu et al, 1999).

Production of recombinant NB-3-His fusion protein. A soluble form ofNB-3 recombinant protein, NB-3- His, was produced in HEK293 cells. Theregion of rat NB-3 cDNA encoding the signal sequence of the GPI-anchorwas substituted with 6×His followed by a termination codon. The NB-3cDNA thus manipulated was inserted into pREP4 between Hind III and BamHIsites, and the resulting vector was transfected into HEK293 cells. Whenthe cells expressing NB-3-His reached confluence they were cultured inserum-free medium for a week. The culture medium was collected,concentrated and dialyzed against 50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0.Ni-NTA resin (Qiagen) was added to the dialysate and incubated for atleast 30 min. NB-3 recombinant protein was eluted from the Ni-NTA resinusing 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole.

Production of Notch1 GST fusion proteins. Production of Notch1 GSTfusion proteins has been described (Hu et al, 2003). Recombinantproteins comprising different regions of the extracellular domain ofmouse Notch1 (mN1) were produced as follows. Primers with added Hind IIIand BspE1 sites (underlined below) were used to amplify regions of mNlcDNA (gift from Dr. Jeffrey Nye) in polymerase chain reactions. N1.1forward :5′-GGTGGAATTCTAATGCCACGGCTCCTG-3′ reverse:5′-TTGAAGTTCCTCATCCGTGTTGATTT-3′ N1.2 forward:5′-TGTGGAATTCTATGTGATCTGGGTGCC-3′ reverse:5′-CGTCAAGTTCGTCATCGATGTCACTCT-3′ N1.3 forward:5′-CTTGGAATTCTATGTGCTACCAGCCCC-3′ reverse:5′-TTGAAGCTTGCCATTGATGACTGACT-3′ N1.4 forward:5′-ACTGGAATTCTATGCCATCCCCCCCTT-3′ reverse:5′-AAGGAAGCTTCTGCGAGGGCAGCGGAG-3′

The regions amplified were N1.1 (nucleotides 79-1557; 1478 bp fragmentencoding amino acids 27-519; EGF repeats 1-13), N1.2 (nucleotides1324-2808; 1484 bp fragment encoding amino acids 442-936; EGF repeats11-24), N1.3 (nucleotides 2575-4008; 1433 bp fragment encoding aminoacids 859-1336; EGF repeats 22-34), and N1.4 (nucleotides 3751-5247;1496 bp fragment encoding amino acids 1251-1749; EGF repeats 32-36, LNRrepeats). Recombinant GST fusion proteins were produced using pGEX-KGvector and purified as described (Xiao et al, 1996).

The PCR was performed on plasmid DNA in the presence of 1 mM MgCl₂. Thecycles were: first five cycles at 93° C. for 1 min, 45° C. for 30 s and72° C. for 1 min; the subsequent 30 cycles were carried out at 93° C.for 1 min, 50° C. for 30s and 72° C. for 1 min; and a final extension at72° C. for 5 min. These amplified fragments were respectively used tocreate vectors pN1.1, pN1.2, pN1.3, and pN1.4 by restriction at at theirextended HindIII and BspEI sites and ligation into similarly digestedPGEX-KG vector. Transformed E. coli JM 109 cells were induced with IPTG,and the expressed GST-N1.1, -N1.2, -N1.3, and -N1.4 fusion proteins werepurified using glutathione-agarose beads.

Production of recombinant NB-3-Fc proteins. The signal sequence of theGPI-anchor was substituted with human IgG Fc followed by a terminationcodon. The cDNA thus manipulated was inserted into pREP4 between HindIII and BamHI sites, and transfected into 293T cells. NB-3-Fc waspurified from the conditioned medium using Protein A-Agarose beads(Roche).

Immunohistochemistry

Following deep anaesthesia, adult Wistar rats were transcardiallyperfused with Ringer's saline followed by 4% paraformaldehyde in 0,1Mphosphate buffer (PB; pH 7.4). Brain stem segments were harvested andpost-fixed for 2 hours before being transferred into 0.1M PB containing15% sucrose overnight. The tissue was frozen in O.C.T. compound(Tissue-Tek) and sagittal cryosections (10 μm) were collected ongelatin-coated slides. For immunostaining, the slides were dried at 37°C. for 30 min, then immersed in cold acetone (−20° C.) for 15 minutesfor permeabilization. Blockade of non-specific binding sites was carriedout using 10% goat serum solution in 0.1M PB for 30 minutes at roomtemperature. Between steps involving antibodies, preparations werewashed three times for 5 minutes each with 0.3% Triton X-100 in 0.1MPBS. All antibodies were diluted in 0.1M PB. Sections were doublelabeled. Tissue sections were typically first incubated overnight withthe polyclonal primary antibody, followed by a secondary goatanti-rabbit antibody. Sections were then exposed to the monoclonalprimary antibody and labeled with an anti-mouse secondary antibody. Forexamination, coverslips were applied and the slides viewed on a Zeisslaser scanning confocal microscope.

Co-Immunoprecipitation and GST Pull-Down Assay

Rat brain membrane samples were prepared as described (Xiao et al, 1996)and incubated overnight at 4° C. with antibody-coupled Protein A agarosebeads (Roche) or glutathione-agarose beads (Sigma) bound to GST-N1.1,N1.2, N1.3 or N1.4. Captured proteins were eluted from beads withSDS-PAGE sample buffer, subjected to Western blot and detection usingECL reagent (Amersham).

NSCs and Rat brain membrane portions were prepared as described (Xiao etal, 1996) and incubated overnight at 4° C. with NB-3-Fc andantibody-coupled Protein A-Agarose beads. Captured proteins were elutedfrom beads with SDS-PAGE sample buffer, subjected to Western blot anddetection using ECL reagent.

Immunoprecipitation of Caspr. Chick or mouse brain lysate (800 μg),prepared as described (Zeng et al, 1999), was incubated with rabbitpre-immune serum (PI) or anti-Caspr serum followed by ProteinA+G-Sepharose (Sigma). The immunoprecipitates were washed, resolevd bySDS-PAGE and probed with anti-Caspr serum. For immunoblotting,pre-immune and immune sera were used at a 1:1000 dilution.

Co-immunoprecipitation. Brain membranes were prepared as describedpreviously (Isom et al, 1995). Membranes were solubilized in 2% TritonX-100 and the soluble fraction was incubated overnight at 4° C. withNotch antibodies (anti-Notch1 and anti-Notch2). Protein A Sepharosebeads (50 μl of a 1:1 suspension) were then added and the incubationcontinued for a further 2 hours or overnight at 4° C. The beads werewashed with 50 mM Tris HCL, pH 7.5, containing 0.1% Triton X-100 andprotease inhibitors. Immunoprecipitated proteins were eluted from thebeads with SDS-PAGE sample buffer and separated on 7.5% acrylamideSDS-PAGE gels. Proteins were transferred to nitrocellulose and probedwith antibodies against F3 and NB-3. Chemiluminescent detection ofimmunoreactive bands was accomplished with ECL reagent. The reciprocalexperiment was carried out using anti-Notch1 and anti-Notch2 antibodiesto probe immunoprecipitates obtained using anti-F3 and anti-NB-3.

GST Pull Down Assay. Purified GST fusion proteins (mNotch 1.1, 1.2, 1.3and 1.4) were coupled to Sepharose 4B beads (Amersham Pharmacia BiotechInc) according to the manufacturer's instructions. Fresh cerebralhemispheres of adult rats were harvested and were solubilized in 2%Triton X-100. The homogenate was centrifuged at 13,000 g for 60 minutes.The cleared lysate was then incubated for 45 min at room temperaturewith GST fusion protein bound to the beads. The beads were washed 3times with lysis buffer and proteins were eluted with SDS-PAGE gelsample buffer, resolved on 7.5% SDS-PAGE, transferred to nitrocelluloseand analyzed by Western blotting using anti-F3 and anti-NB-3 antibodies.

Culture of Primary OLS and OPCs

The inventor employed a glial cell separation technique in whicholigodendrocytes are separated by Percoll gradient centrifugation aspreviously described (Colello and Sato-Bigbee, 1998). Briefly, an hourbefore plating cells, culture dishes (Falcon) were coated withpoly-L-lysine. Postnatal day 1-2 rats were sacrificed by decapitationand their cerebral hemispheres rapidly dissected out. Meninges and bloodvessels were carefully teased away using microdissecting forceps.Hemispheres were transferred to ice-cold HEPES/HBSS in a petri dish andfinely minced using a scalpel blade. The tissue was transferred to a 50ml conical tube and centrifuged for 5 minutes at 200×g after which thesupernatant was discarded and the cell pellet resuspended in 10 μg/mlDNAase I in HEPES/HBSS before subjecting it to centrifugation again. Asingle cell suspension was prepared by forcing the tissue through a 70μm nylon mesh Falcon cell strainer, applied to a Percoll gradient, andthe oligodendrocyte-containing fraction recovered after centrifugation.The cell suspension was diluted with HEPES/HBSS and oligodendrocyteswere separated by differential adhesion. Oligodendrocytes obtained inthis manner were suspended in chemically defined DMEM/ F12 medium.

Culture of primary neurons, Ols, and astrocytes. Neurons, OLs, andastrocytes of E17 Wistar rats were isolated and cultured as described(Itoh, 2002). OLs of P5-7 Wistar rat cerebella were obtained by Percollgradient centrifugation (Colello and Sato-Bigbee, 1998) and OPCs werepurified from P5-7 Wistar rat optic nerve (Bogler, 1997).

Culture of NSCs. Murine striatal neural stem cells were isolated from14-d-old BALB/c mouse embryos (IFFA Credo, L'Arbresle, France) andcultured in DMEM/F12 with N2 supplement and EGF (20 ng/ml) (Invitogen)(Arsenijevic et al, 2001). To induce differentiation, the spheres weremechanically dissociated into single cells and treated with NB-3 orJagged1 in culture medium without growth factor for 24 hrs, and thenadded into culture medium with 1% fetal calf medium.

Real-Time RT-PCR Analysis

Primary oligodendrocytes were plated onto protein spots of F3-Fc,NB-3-His and BSA in a similar fashion to the cell adhesion assaypreviously described. After 2 hours in culture, total RNA was extractedaccording to the manufacturer's instructions using a QIAGEN RNAeasy kit.Total RNA (0.5 μg) was reverse transcribed using the TaqMan RT Kit(Applied Biosystems). To measure the level of mRNA expression, weperformed real-time quantitative PCR using the TaqMan system on an ABIPRISM 7700 Sequence Detection System. All primers and probes weredesigned using Primer Express Software (ABI). Primer and probe sequencesare as follows. Myelin Associated Glycoprotein (MAG) Forward Primer:5′-ATCCTGGCCACGGTCATC-3′ Reverse Promer: 5′-CACACCAGTACTCCCCATCGT-3′Taqman Probe: 5′-CAGCTGGAACTCCCTGCAGTGACG-3′ Proteolipid Protein (PLP)Forward Primer: 5′-AGGCCAACATCAAGCTCATTCT-3′ Reverse Primer:5′-CGGGATGTCCTAGCCATTTTC-3′ Taqman Probe: 5′-CCAAACAATGACACACCCGCTCCA-3′

Independent amplifications of each target and the GAPDH RNA wereperformed according to the manufacturer's instructions. Relativeexpression levels of each transcript were determined by employing thecomparative C_(T) method as outlined in the ABI User's manual.

Total RNA from OLs or OLN cells was extracted using the QIAGEN RNeasykit and treated with RNase-free DNaseI (Invitrogen) to eliminate genomicDNA. Samples were used for reverse transcription with random hexamerprimers using SuperScript First-Strand Synthesis System (Invitrogen)(Notch homologs, Hes1) or TaqMan RT Kit (Applied Biosystems) (MAG).β-actin and GAPDH were used as internal controls. Real-time PCR wasperformed using the SYBR Green PCR Master Mix (Notch homologs, Hes1) orTaqMan system (MAG) on an ABI PRISM 7700 Sequence Detection System. Theprimers and TaqMan probes were designed using Primer Express Software(ABI) and sequences are available upon request. The raw data from atleast four independent experiments were used to determine the relativeexpression levels of each transcript by employing the comparative C_(T)method (ABI User's manual).

Lipid Raft Assay

This was performed as described (Krämer et al, 1999).

Hes1 Luciferase Reporter Assay

OLN cells (1.5×10⁵/well) in 12-well dishes were used for Hes1 luciferasereporter assays. Cells were transiently transfected using Lipofectamineand Lipofectamine Plus reagents (Invitrogen). Each well received 0.2 μgpGVB/Hes1 luciferase reporter plasmid together with various expressionplasmids (0.1 or 0.2 μg caN1; 0.3 or 0.6 μg RBP-J, dn-RBP-J-myc, DTX1,DTX1-D1-HA, DTX1-D2-Flag). The β-galactosidase expression plasmidpCMV/β-Gal was included as internal control to monitor the transfectionefficiency. Cells were lysed 24 hours post-transfection and assayedusing the Steady-Glo Luciferase Assay Kit (Promega). The raw data fromat least four independent experiments were used to determine therelative reporter activity.

The NSC experiments was performed as described (Hu et al, 2003). Inbrief, NSCs in 24-well dishes were transiently transfected withindicated constructs using Lipofectamine 2000 (Invitrogen). pCMV/β-Galexpressing β-galactosidase was cotransfected to monitor the transfectionefficiency. Cells were subjected to luciferase assay 24 hourspost-transfection using the Steady-Glo Luciferase Assay Kit (Promega).The raw data from at least four independent experiments were used todetermine the relative reporter activity.

Flow Cytometric Analysis

The cells were trypsinized, washed with PBS and treated with FACSPerm(Becton Dickinson). Cells were stained with antibody to MAP2 (2a+2b),GFAP, CNPase and FITC-conjugated anti-mouse IgG1 and anti rabbit IgG,then analyzed by flow cytometer (FACScalibur, Becton Dickinson) withCELLQuest software Version (Becton Dickinson).

REFERENCES FOR THE FIRST ASPECT OF THE INVENTION

-   Ahn, M., D. S. Min, J. Kang, K. Jung, and T. Shin. 2001. Increased    expression of phospholipase Dl in the spinal cords of rats with    experimental autoimmune encephalomyelitis. Neurosci. Lett.    316:95-98.-   Ang, B. T., M. Karsak, S. Lee, Y. Takeda, U. Lendahl, G. Rougon, A.    Israel, M. Schachner, K. Watanabe, and Z. C. Xiao. 2001. Notch is a    receptor for F3/contactin, and NB-3: a paranodal axon-glial    signaling mechanism during myelination. Soc. Neurosci. Abst. 900.5-   Barres, B. A., and M. C. Raff. 1999. Axonal control of    oligodendrocyte development. J. Cell Biol. 147:1123-1128.-   Bhat, M. A., J. C. Rios, Y. Lu, G. P. Garcia-Fresco, W. Ching, M. St    Martin, J. Li, S. Einheber, M. Chesler, J. Rosenbluth, J. L. Salzer,    and H. J. Bellen. 2001. Axon-glia interactions and the domain    organization of myelinated axons requires neurexin    IV/Caspr/Paranodin. Neuron. 30:369-383.-   Boyle, M. E., E. O. Berglund, K. K. Murai, L. Weber, E. Peles,    and B. Ranscht. 2001. Contactin orchestrates assembly of the    septate-like junctions at the paranode in myelinated peripheral    nerve. Neuron. 30:385-97.-   Brittis, P. A., and J. G. Flanagan. 2001. Nogo domains and a Nogo    receptor: implication for axon regeneration. Neuron. 30:11-14.-   Buttiglione, M, J. M. Revest, O. Pavlou, D. Karagogeos, A.    Furley, G. Rougon, and C. Faivre-Sarrailh. 1998. A functional    interaction between the neuronal adhesion molecules TAG-1 and F3    modulates neurite outgrowth and fasciculation of cerebellar granule    cells. J Neurosci. 18: 6853-6870.-   Charles, P., S. Tait, C. Faivre-Sarrailh, G. Barbin, F.    Gunn-Moore, N. Denisenko-Nehrbass, A. M. Guennoc, J. A.    Girault, P. J. Brophy, and C. Lubetzki. 2002. Neurofascin is a glial    receptor for the paranodin/Caspr-contactin axonal complex at the    axoglial junction. Curr. Biol. 12:217-220.-   Chen, M. S., A. B. Huber, M. E. van der Haar, M. Frank, L.    Schnell, A. A. Spillmann, F. Christ, and M. E. Schwab. 2000. Nogo-A    is a myelin-associated neurite outgrowth inhibitor and an antigen    for monoclonal antibody IN-1. Nature. 403:434-439.-   Coetzee, T., Fujita, N., Dupree, J., Shi, R., Blight, A., Suzuki,    K., Suzuki, K., Popko, B. 1996. Myelination in the absence of    galactocerebroside and sulfatide: normal structure with abnormal    function and regional instability. Cell. 86(2): 209-219.-   Dawson, M. R., Levine, J. M., and Reynolds, R. 2000. NG2-expressing    cells in the central nervous system: are they oligodendroglial    progenitors? J. Neurosci. Res. 61, 471-479.-   Dupree, J. L., J. A. Girault, and B. Popko. 1999. Axo-glial    interactions regulate the localization of axonal paranodal    proteins. J. Cell Biol. 147:1145-1152.-   Einheber, S., G. Zanazzi, W. Ching, S. Scherer, T. A. Milner, E.    Peles, and J. L. Salzer. 1997. The axonal membrane protein Caspr, a    homologue of neurexin IV, is a component of the septate-like    paranodal junctions that assemble during myelination. J. Cell Biol.    139:1495-1506.-   Faivre-Sarrailh, C., F. Gauthier, N. Denisenko-Nehrbass, A. Le    Bivic, G. Rougon, and J A. Girault. 2000. The glycosylphosphatidyl    inositol-anchored adhesion molecule F3/contactin is required for    surface transport of paranodin/contactin-associated protein    (caspr). J. Cell Biol. 149:491-502.-   Fournier, A. E., T. GrandPre, and S. M. Strittmatter. 2001.    Identification of a receptor mediating Nogo-66 inhibition of axonal    regeneration. Nature. 409:341-346.-   Gennarini, G., P. Durbec, A. Boned, G. Rougon, and C. Goridis. 1991.    Transfected F3/F11 neuronal cell surface protein mediates    intercellular adhesion and promotes neurite outgrowth. Neuron.    6:595-606.-   Girault, J. A., and Peles, E. 2002. Development of nodes of Ranvier.    Curr Opin Neurobiol. 12(5):476-485.-   Gollan, L., H. Sabanay, S. Poliak, E. O. Berglund, B. Ranscht,    and E. Peles. 2002. Retention of a cell adhesion complex at the    paranodal junction requires the cytoplasmic region of Caspr. J. Cell    Bio. 157:1247-1256-   GrandPre, T., F. Nakamura, T. Vartanian, and S. M.    Strittmatter. 2000. Identification of the Nogo inhibitor of axon    regeneration as a reticulon protein. Nature. 403:439-444.-   Guan, K. L., and J. E. Dixon. 1991. Eukaryotic proteins expressed in    Escheridhia coli: an improved thrombin cleavage and purification    procedure of fusion proteins with glutathione S-transferase. Anal.    Biochem. 192:262-267.-   Hu, W. H., O. N. Hausmann, M. S. Yan, W. M. Walters, P. K. Wong,    and J. R. Bethea. 2002. Identification and characterization of a    novel Nogo-interacting mitochondrial protein (NIMP). J. Neurochem.    81:36-45.-   Huber, A. B., O. Weinmann, C. Brosamle, T. Oertle, and M. E.    Schwab. 2002. Pattern of Nogo mRNA and protein expression in the    developing and adult rat and after CNS lesions. J. Neurosci.    22:3553-3567.-   Hunt, D., M. Mason, G. Campbell, R. Coffin, and P. Anderson. 2002.    Nogo receptor mRNA expression in intact and regenerating CNS    neurons. Mol. Cell. Neurosci. 20:537.-   Ishibashi, T., J. L. Dupree, K. Ikenaka, Y. Hirahara, K. Honke, E.    Peles, B. Popko, K. Suzuki, H. Nishino, and H. Baba. 2002. A myelin    galactolipid, sulfatide, is essential for maintenance of ion    channels on myelinated axon but not essential for initial cluster    formation. J. Neurosci. 22:6507-6514.-   Kazarinova-Noyes, K., J. D. Malhotra, D. P. McEwen, L. N.    Mattei, E. O. Berglund, B. Ranscht, S. R. Levinson, M. Schachner, P.    Shrager, L. L. Isom, and Z. C. Xiao. 2001. Contactin associates with    Na+ channels and increases their functional expression. J. Neurosci.    21:7517-7525.-   Krämer, E. M., C. Klein, T. Koch, M. Boytinck, and J. Trotter. 1999.    Compartmentation of Fyn kinase with    glycosylphosphatidylinositol-anchored molecules in oligodendrocytes    facilitates kinase activation during myelination. J. Biol. Chem.    274:29042-29049.-   Lee, S., Y. Takeda, H. Kawano, H. Hosoya, M. Nomoto, D. Fujimoto, N.    Takahashi, and K. Watanabe. 2000. Expression and regulation of a    gene encoding neural recognition molecule NB-3 of the contactin/F3    subgroup in mouse brain. Gene. 245:253-66.-   Lei, G., Xue, S., Chéry, N., Liu, Q., Xu, J., Kwan, C. L., Fu, Y.    P., Lu, Y. M., Liu, M., Harder, K. W., Yu, X. M. 2002. Gain control    of N-methyl-D-aspartate receptor activity by receptor-like protein    tyrosine phosphatase α. EMBO J. 21(12): 2977-2989.-   Liu, H., C. E. Ng, and B. L. Tang. 2002. Nogo-A expression in mouse    central nervous system neurons. Neurosci. Lett. 328:257-260.-   Marcus, J., J. L. Dupree, and B. Popko. 2000. Effects of    galactolipid elimination on oligodendrocyte development and    myelination. Glia. 30:319-328.-   Menegoz, M., P. Gaspar, M. Le Bert, T. Galvez, F. Burgaya, C.    Palfrey, P. Ezan, F. Arnos, and J. A. Girault. 1997. Paranodin, a    glycoprotein of neuronal paranodal membranes. Neuron. 19:319-331.-   Nakahira, K., Shi, G., Rhodes, K. J., Trimmer, J. S. 1996. Selective    interaction of voltage-gated K⁺ channel β-subunits with    α-subunits. J. Bio. Chem. 271(12): 7084-7089.-   Pedraza, L., J. K. Huang, and D. R. Colman. 2001. Organizing    principles of the axoglial apparatus. Neuron. 30:335-344.-   Peles, E., K. Joho, G. D. Plowman, and J. Schlessinger. 1997. Close    similarity between Drosophila neurexin IV and mammalian Caspr    protein suggests a conserved mechanism for cellular interactions.    Cell. 88:745-746.-   Poliak, S., L. Gollan, R. Martinez, A. Custer, S. Einheber, J. L.    Salzer, J. S. Trimmer, P. Shrager, and E. Peles. 1999. Caspr2, a new    member of the neurexin superfamily, is localized at the    juxtaparanodes of myelinated axons and associates with K⁺ channels.    Neuron. 24:1037-47.-   Poliak, S., Gollan, L., Salomon, D., Berglund, E. O., Ohara, R.,    Ranscht, B., Peles, E. 2001. Localization of Caspr2 in myelinated    nerves depends on axon-glia interactions and the generation of    barriers along the axon. J Neurosci. 21(19):7568-7575.-   Popko B. 2000. Myelin galactolipids: mediators of axon-glial    interactions? Glia. 29:149-153.-   Prinjha, R., S. E. Moore, M. Vinson, S. Blake, R. Morrow, G.    Christie, D. Michalovich, D. L. Simmons, and F. S. Walsh. 2000.    Inhibitor of neurite outgrowth in humans. Nature. 403:383-384.-   Rasband, M. N., and P. Shrager. 2000. Ion channel sequestration in    central nervous system axons. J. Physio. 525:63-73.-   Rasband, M. N., and J. S. Trimmer. 2001a. Developmental clustering    of ion channels at and near the Node of Ranvier. Dev. Biol.    236:5-16.-   Rasband, M. N., and J. S. Trimmer. 2001b. Subunit composition and    novel localization of K⁺ channels in spinal cord. J. Comp. Neurol.    429: 166-176.-   Rasband, M. N., J. S. Trimmer, E. Peles, S. R. Levinson, and P.    Shrager. 1999. K⁺ channel distribution and clustering in developing    and hypomyelinated axons of the optic nerve. J. Neurocytol.    28:319-331.-   Rasband, M. N., J. S. Trimmer, T. L. Schwarz, S. R. Levinson, M. H.    Ellisman, M. Schachner, and P. Shrager. 1998. Potassium channel    distribution, clustering, and function in remyelinating rat    axons. J. Neurosci. 18:36-47.-   Scherer, S. S., and E. J. Arroyo. 2002. Recent progress on the    molecular organization of myelinated axons. J. Peripher. Nerv. Syst.    7:1-12.-   Spiegel, I., D. Salomon, B. Erne, N. Schaeren-Wiemers, and E.    Peles. 2002. Caspr3 and caspr4, two novel members of the caspr    family are expressed in the nervous system and interact with PDZ    domains. Mol. Cell. Neurosci. 20:283-297.-   Swanborg, R. H. 2001. Experimental autoimmune encephalomyelitis in    the rat: lessons in T-cell immunology and autoreactivity. Immunol    Rev. 184: 129-135.-   Taketomi, M., N. Kinoshita, K. Kimura, M. Kitada, T. Noda, H.    Asou, T. Nakamura, and C. Ide. 2002. Nogo-A expression in mature    oligodendrocytes of rat spinal cord in association with specific    molecules. Neurosci. Lett. 332:37.-   Tait, S., F. Gunn-Moore, J. M. Collinson, J. Huang, C. Lubetzki, L.    Pedraza, D. L. Sherman, D. R. Colman, and P. J. Brophy. 2000. An    oligodendrocyte cell adhesion molecule at the site of assembly of    the paranodal axo-glial junction. J. Cell Biol. 150:657-666.-   Trimmer, J. S. 1991 Immunological identification and    characterization of a delayed rectifier K+ channel polypeptide in    rat brain. Proc Natl Acad Sci USA. 88(23):10764-10768.-   Vabnick, I., and Shrager, P. 1998. Ion channel redistribution and    function during development of the myelinated axon. J Neurobiol.    37(1):80-96.-   Vabnick, I., J. S. Trimmer, T. L. Schwarz, S. R. Levinson, D. Risal,    and P. Shrager. 1999. Dynamic potassium channel distributions during    axonal development prevent aberrant firing patterns. J. Neurosci.    19:747-758.-   Wang, X., S. J. Chun, H. Treloar, T. Vartanian, C. A. Greer,    and S. M. Strittmatter. 2002. Localization of Nogo-A and Nogo-66    receptor proteins at sites of axon-myelin and synaptic contact. J.    Neurosci. 22:5505-5515.-   Woolf, C. J. 2003. No Nogo: now where to go? Neuron. 38(2):153-156-   Xiao, Z. C., J. Taylor, D. Montag, G. Rougon, and M.    Schachner. 1996. Distinct effects of tenascin-R domains in neuronal    cell functions and identification of the domain interacting with the    neuronal recognition molecule F3/F11. Eur. J. Neurosci. 8:766-782.

REFERENCE FOR THE SECOND ASPECT OF THE INVENTION

-   Arsenijevic, Y., Weiss, S., Schneider, B. & Aebischer, P. 2001. J.    Neurosci. 21, 7194-7202.-   Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J. 1999. Notch    signalling: cell fate control and signal integration in development.    Science 284, 770-776.-   Arroyo, E. J. and Scherer, S. S. 2000. On the molecular architecture    of myelinated fibres. Histochem. Cell Biol. 113, 1-18.-   Baker, N. E. 2000. Notch signalling in the nervous system. Pieces    still missing from the puzzle. BioEssays 22, 264-273.-   Barres, B. A. & Raff, M. C. 1999. Axonal control of oligodendrocyte    development. J. Cell Biol. 147, 1123-1128-   Baumgartner, S., Littleton, J. T., Broadie, K., Bhat, M. A.,    Harbecke, R., Lengyel, J. A., Chiquet-Ehrismann, R., Prokop, A. and    Bellen, H. J. 1996. A drosophila neurexin is required for septate    junction and blood-nerve barrier formation and function. Cell 87,    1059-1068.-   Berglund, E. O., Murai, K. K., Fredette, B., Serkekova, G.,    Marturano, B., Weber, L., Mugnaini, E. and Ranscht, B. 1999 Ataxia    and abnormal cerebellar microorganization in mice with ablated    contactin gene expression. Neuron 24, 739-750.-   Bogler, O. 1997. Isolation and purification of primary    oligodendrocyte precursors. In Current Protocols in Neuroscience.    3.4.1-3.4.9 (John Wiley & Sons, Inc).-   Bosio, A., Bussow, H., Adam, J. and Stoffel, W. 1998.

Galactosphingolipids and axonal-glial interaction in myelin of thecentral nervous system. Cell Tissue Res. 292, 199-210.

-   Boyle, M. E. T., Berglund, E. O., Murai, K. K., Weber, L., Peles, E.    and Ranscht, B. 2001. Contactin orchestrates assembly of the    septate-like junctions at the paranode in myelinated peripheral    nerve. Neuron 30, 385-387.-   Brennan, K., and Gardner, P. 2002. Notching up another pathway.    Bioessays 24, 405-410.-   Coetzee, T., Fujita, N., Dupree, J., Shi, R., Blight, A., Suzuki, K.    and Popko, B. 1996. Myelination in the absence of galactocerebroside    and sulfatide: normal structure with abnormal function and regional    instability. Cell 86, 209-219.-   Colello, R. J. and Sato-Bigbee, C. 1998. Purification of    oligodendrocytes and their progenitors using immunomagnetic    separation and percoll gradient centrifugation. In: Current    Protocols in Neuroscience (Crawley, J. N. et. al. eds.). John Wiley    and Sons, Inc., pp. 3.12.7-3.12.10.-   Dale, J. K., and Maroto, M. 2003. A HES1-based oscillator in    cultured cells and its potential implications for the segmentation    clock. Bioessays 25, 200-203.-   Dawson, M. R., Levine, J. M., and Reynolds, R. 2000. NG2-expressing    cells in the central nervous system: are they oligodendroglial    progenitors? J. Neurosci. Res. 61, 471-479-   Denisenko-Nehrbass, N., Faivre-Sarrailh, C., Goutebroze, L. and    Girault, J.-A. 2002. A molecular view on paranodal junctions of    myelinated fibres. J. Physiology (Paris) 96, 99-103.-   Dugas, J. C., Milligan, B. D. and Barres, B. A. 2001. Onset of    myelination is triggered by target innervation. Soc. Neurosci. Abst.    900.4.-   Ebinu, J. O., and Yankner, B. A. 2002. A RIP tide in neuronal signal    transduction. Neuron 34, 499-502.-   Einheber, S., Zanazzi, G., Ching, W., Scherer, S., Milner, T. A.,    Peles, E. and Salzer, J. L. 1997. The axonal membrane protein Caspr,    a homologue of neurexin IV, is a component of the septate-like    paranodal junctions that assemble during myelination. J. Cell Biol.    139, 1495-1506.-   Ellisen, L. W., Bird, J., West, D. C., Soreng, A. L., Reynolds, T.    C., Smith, S. D. and Sklar, J. 1991. TAN-1, the human homolog of the    drosophila Notch gene, is broken by chromosomal translocations in T    lymphoblastic neoplasms. Cell 66, 649-661.-   Faivre-Sarrailh, C., Gauthier, F., Denisenko-Nehrbass, N., Le Bivic,    A., Rougon, G. and Girault, J. A. 2000. The glycosylphosphatidyl    inositol-anchored adhesion molecule F3/contactin is required for    surface transport of paranodin/contactin-associated protein    (caspr). J. Cell Biol. 149, 491-502.-   Furley, A. J., Morton, S. B., Manalo, D., Karagogeos, D., Dodd, J.    and Jessell, T. M. 1990. The axonal glycoprotein TAG-1 is an    immunoglobulin superfamily member with neurite outgrowth-promoting    activity. Cell 61, 157-170.-   Furukawa, T., Mukherjee, S., Bao, Z. Z., Morrow, E. M., and    Cepko, C. L. 2000. rax, HES1, and Notch1 promote the formation of    Muller glia by postnatal retinal progenitor cells. Neuron 26,    383-394.-   Gaiano, N., Nye, J. S. and Fischell, G. 2000. Radial glial identitiy    is promoted by Notch1 signalling in the murine forebrain. Neuron 26,    395-404.-   Gennarini, G., Cibelli, G., Rougon, G., Mattei, M. G. and    Gorodis, C. 1989. The mouse neuronal cell surface protein F3: a    phosphatidyl-inositol anchored member of the immunoglobulin    superfamily related to chicken contactin. J. Cell Biol. 109,    775-788.-   Gennarini, G., Durbec, P., Boned, A., Rougon, G. and    Goridis, C. 1991. Transfected F3/F11 neuronal cell surface protein    mediates intercellular adhesion and promotes neurite outgrowth.    Neuron 6, 595-606.-   Genoud, S., Lappe-Siefke, C., Goebbels, S., Radtke, F., Aguet, M.,    Scherer, S. S., Suter, U., Nave, K. A., and Mantei, N. 2002. Notch1    control of oligodendrocyte differentiation in the spinal cord. J.    Cell Biol. 158, 709-18.-   Girault, J. A., and Peles, E. 2002. Development of nodes of Ranvier.    Curr. Opin. Neurobiol. 12, 476-485.-   Guan, K. L. and Dixon, J. E. 1991. Eukaryotic proteins expressed in    Escherichia coli: an improved thrombin cleavage and purification    procedure of fusion proteins with glutathione-S-transferase. Anal.    Biochem. 192, 262-267.-   Harlow, Ed., David, L. 1998. Immunizations. In Antibodies A    Laboratory Manual. 53-244 (Cold Spring Harbor Laboratory).-   Hemann, C., Gartner, E., Weidle, U. H. and Grummt, F. 1994.    High-copy expression vector based on amplification-promoting    sequences. DNA Cell Biol. 13, 437-445.-   Hirata, H., Yoshiura, S., Ohtsuka, T., Bessho, Y., Harada, T.,    Yoshikawa, K., and Kageyama, R. 2002. Oscillatory expression of the    bHLH factor Hes1 regulated by a negative feedback loop. Science 298,    840-843.-   Hojo, M., Ohtsuka, T., Hashimoto, N., Gradwohl, G., Guillemot, F.,    and Kageyama, R. 2000. Glial cell fate specification modulated by    the hHLH gene HES5 in mouse retina. Development 127, 2515-2522.-   Holm, J., Hillenbrand, R., Steuber, V., Bartsch, U., Moos, M.,    Lubbert, H., Montag, D. and, Schachner, M. 1996. Structural features    of a close homologue of L1 (CHL1) in the mouse: a new member of the    L1 family of neural recognition molecules. Eur. J. Neurosci. 8,    1613-29.-   Hoover, K. B. and Bryant, P. J. 2000. The genetics of the protein    4.1 family: organizers of the membrane and cytoskeleton. Curr. Opin.    Cell Biol. 12, 229-234.-   Hu, Q. D., Ang, B. T., Karsak, M., Hu, W. P., Cui, X. Y., Duka, T.,    Takeda, Y., Chia, W., Natesan, S., Ng, Y. K., Ling, E. A., Maciag,    T., Small, D., Trifonova, R., Kopan, R., Okano, H., Nakafuku, M.,    Chiba, S., Hirai, H., Aster, J. C., Schachner, M., Pallen, C. J.,    Watanabe, K., and Xiao, Z. C. 2003. F3/Contactin acts as a    functional ligand for Notch during oligodendrocyte differentiation.    Cell (in press).-   Huppert, S. S., Le, A., Schroeter, E. H., Mumm, J. S., Saxena, M.    T., Milner, L. A., and Kopan, R. 2000. Embryonic lethality in mice    homozygous for a processing-deficient allele of Notch1. Nature 405,    966-970.-   Isom, L. L., Ragsdale, D. S., De Jongh, K. S., Westenbroek, R. E.,    Reber, B. F., Scheuer, T. and Catterall, W. A. 1995. Structure and    function of the beta 2 subunit of brain sodium channels, a    transmembrane glycoprotein with a CAM motif. Cell 83, 433-442.-   Itoh, K. 2002. Culture of oligodendrocyte precursor cells (NG2+/O1−)    and oligodendrocytes (NG2−/O1+) from embryonic rat cerebrum. Brain    Res. Brain Res. Protoc. 10, 23-30.-   Izon, D. J., Aster, J. C., He, Y., Weng, A., Karnell, F. G.,    Patriub, V., Xu, L., Bakkour, S., Rodriguez, C., Allman, D., and    Pear, W. S. 2002. Deltex1 redirects lymphoid progenitors to the B    cell lineage by antagonizing Notch1. Immunity 16, 231-243.-   Jack, C., Berezovska, O., Wolfe, M. S., and Hyman, B. T. 2001.    Effect of PS1 deficiency and an APP gamma-secretase inhibitor on    Notch1 signaling in primary mammalian neurons. Brain Res. Mol.    Brain. Res. 87, 166-174.-   John, G. R., Shankar, S. L., Shafit-Zagardo, B., Massimi, A.,    Lee, S. C., Raine, C. S., and Brosnan, C. F. 2002. Multiple    sclerosis: Re-expression of a developmental pathway that restricts    oligodendrocyte maturation. Nat. Med. 8, 1115-1121.-   Joutel, A., Vahedi, K., Corpechot, C., Troesch, A., Chabriat, H.,    Vayssiere, C., Cruaud, C., Maciazek, J., Weissenbach, J.,    Bousser, M. G., Bach, J. F. and Tournier-Lasserve, E. 1997 Strong    clustering and stereotyped nature of Notch3 mutations in CADASIL    patients. Lancet 350, 1511-1515.-   Jung, M., Sommer, I., Schachner, M. and Nave, K. A. 1996. Monoclonal    antibody O10 defines a conformationally sensitive cell-surface    epitope of proteolipid protein (PLP): evidence that PLP misfolding    underlies dysmyelination in mutant mice. J. Neurosci. 16, 7920-7929.-   Kabos, P., Kabosova, A., and Neuman, T. 2002. Blocking HES1    expression initiates GABAergic differentiation and induces the    expression of p21 (CIP1/WAF1) in human neural stem cells. J. Biol.    Chem. 277, 8763-8766.-   Kaneta, M., Osawa, M., Sudo, K., Nakauchi, H., Farr, A. G., and    Takahama, Y. 2000. A role for pref-1 and HES-1 in thymocyte    development. J. Immunol. 164, 256-264.-   Kato, H., Taniguchi, Y., Kurooka, H., Minoguchi, S., Sakai, T.,    Nomura-Okazaki, S., Tamura, K., and Honjo, T. 1997. Involvement of    RBP-J in biological functions of mouse Notch1 and its derivatives.    Development 124, 4133-4141.-   Kazarinova-Noyes, K., Malhotra, J. D., McEwen, D. P., Mattei, L. N.,    Berglund, E. O., Ranscht, B., Levinson, S. R., Schachner, M.,    Shrager, P., Isom, L. L., and Xiao, Z. C. 2001. Contactin associates    with Na⁺ channels and increase their functional expression. J.    Neurosci. 21, 7517-7525.-   Kishi, N., Tang, Z., Maeda, Y., Hirai, A., Mo, R., Ito, M., Suzuki,    S., Nakao, K., Kinoshita, T., Kadesch, T., Hui, C.,    Artavanis-Tsakonas, S., Okano, H., and Matsuno, K. 2001. Murine    homologs of deltex define a novel gene family involved in vertebrate    Notch signaling and neurogenesis. Int. J. Dev. Neurosci. 19, 21-35.-   Klein, C., Kramer, E. M., Cardine, A. M., Schraven, B., Brandt, R.,    and Trotter, J. 2002. Process outgrowth of oligodendrocytes is    promoted by interaction of fyn kinase with the cytoskeletal protein    tau. J. Neurosci. 22, 698-707.-   Koch, T., Brugger, T., Bach, A., Gennarini, G. and Trotter, J. 1997.    Expression of the immunoglobulin superfamily cell adhesion molecule    F3 by oligodendrocyte-lineage cells. Glia 19, 199-212.-   Kramer, E. M., Klein, C., Koch, T., Boytinck, M., and    Trotter, J. 1999. Compartmentation of Fyn kinase with    glycosylphosphatidylinositol-anchored molecules in oligodendrocytes    facilitates kinase activation during myelination. J Biol Chem. 274,    29042-29049.-   Laemmli, U. K. 1970. Cleavage of structural proteins during the    assembly of the head of bacteriophage T4. Nature 277, 680-685.-   Lagenaur, C. and Lemmon, V. 1987. An L1-like molecule, the 8D9    antigen, is a potent substrate for neurite extension. Proc. Natl.    Acad. Sci. USA 84, 7753-7757.-   Lardelli, M., Dahlstrand, J., and Lendahl, U. 1994. The novel Notch    homologue mouse Notch3 lacks specific epidermal growth    factor-repeats and is expressed in proliferating neuroepithelium.    Mech. Dev. 46, 123-136.-   Lee, S., Takeda, Y., Kawano, H., Hosoya, H., Nomoto, M., Fujimoto,    D., Takahashi, N. and Watanabe, K. 2000. Expression and regulation    of a gene encoding neural recognition molecule NB-3 of the    contactin/F3 subgroup in mouse brain. Gene 245, 253-266.-   Logeat, F., Bessia, C., Brou, C., LeBail, O., Jarriault, S.,    Seidah, N. G. and Israel, A. 1998. The Notch1 receptor is cleaved    constitutively by a furin-like convertase. Proc. Natl. Acad. Sci.    USA 95, 8108-8112.-   Martin-Collinson, J., Marshall, D., Stewart Gillespie, C. and    Brophy, P. J. 1998. Transient expression of neurofascin by    oligodendrocytes at the onset of myelinogenesis: Implications for    mechanisms of axon-glial interaction. Glia 23, 11-23.-   Martinez Arias, A., Zecchini, V., and Brennan, K. 2002.    CSL-independent Notch signaling: a checkpoint in cell fate decisions    during development? Curr. Opin. Genet. Dev. 12, 524-533.-   Mathis, C., Denisenko-Nehrbass, N., Girault, J. A. and    Borrelli, E. 2001. Essential role of oligodendrocytes in the    formation and maintenance of central nervous system nodal regions.    Development. 128, 4881-90.-   Matsuno, K., Ito, M., Hori, K., Miyashita, F., Suzuki, S., Kishi,    N., Artavanis-Tsakonas, S., Okano, H. 2002. Involvement of a    proline-rich motif and RING-H2 finger of Deltex in the regulation of    Notch signaling. Development 129, 1049-1059.-   Menegoz, M., Gaspar, P., Le Bert, M., Galvez, T., Burgaya, F.,    Palfrey, C., Ezan, P., Arnos, F. and Girault, J. A. 1997. Paranodin,    a glycoprotein of neuronal paranodal membranes. Neuron 19, 319-331.-   Mitsiadis, T. A., Lardelli, M., Lendahl, U. and Thesleff, I. 1995.    Expression of Notch 1, 2 and 3 is regulated by    epithelial-mesenchymal interactions and retinoic acid in the    developing mouse tooth and associated with determination of    ameloblast cell fate. J. Cell Biol. 130, 407-18.-   Morell, P. and Quarles, R. H. 1999. Myelin formation, structure and    biochemistry. In: Basic Neurochemistry—molecular, cellular and    biochemical aspects, 6^(th) edition (Siegel, G. J. et. al. eds.).    Lippincott-Raven Publishers, Philadelphia, pp. 69-93.-   Morrison, S. J., Perez, S. E., Qiao, Z., Verdi, J. M., Hicks, C.,    Weinmaster, G. and Anderson, D. J. 2000. Transient Notch activation    initiates an irreversible switch from neurogenesis to gliogenesis by    neural crest stem cells. Cell 101, 499-510.-   Mumm. J. S. and Kopan, R. 2000. Notch signalling: From the outside    in. Dev. Biol. 228, 151-165.-   Pedraza, L., Huang, J. K. and Colman, D. R. (2001) Organizing    principles of the axoglial apparatus. Neuron 30, 335-344.-   Peles, E. and Salzer, J. 2000. Molecular domains of myelinated    axons. Curr. Opinion Cell Biol. 10, 558-565.-   Pluchino, S. et al. 2003. Nature 422, 688-694.-   Rand, M. D., Grimm, L. M., Artavanis-Tsakonas, S., Patriub, V.,    Blacklow, S. C., Sklar, J., and Aster, J. C. 2000. Calcium depletion    dissociates and activates heterodimeric Notch receptors. Mol. Cell.    Biol. 20, 1825-1835.-   Rebay, R. J., Fleming, R. G., Fehon, R. G., Cherbas, L., Cherbas, P.    and Artavanis-Tsakanos, S. 1991. Specific EGF repeats of Notch    mediated interactions with Serrate: Implications for Notch as a    multifunctional receptor. Cell 67, 687-699.-   Revest, J. M., Faivre-Sarrailh, C., Schachner, M., and    Rougon, G. 1999. Bidirectional signaling between neurons and glial    cells via the F3 neuronal adhesion molecule. Adv. Exp. Med. Biol.    468, 309-318.-   Richter-Landsberg, C. and Heinrich, M. 1996. OLN-93: A new permanent    oligodendroglia cell line derived from primary rat brain glial    cultures. J. Neurosci. Res. 45, 161-173.-   Rios, J. C., Melendez-Vasquez, C. V., Einheber, S., Lustig, M.,    Grumet, M., Hemperly, J., Peles, E. and Salzer, J. L. 2000.    Contactin-associated protein (Caspr) and contactin form a complex    that is targeted to the paranodal junctions during myelination. J.    Neurosci. 20, 8354-8364.-   Robey, E., Chang, D., Itano, A., Cado, D., Alexander, H., Lans, D.,    Weinmaster, G. and Salmon, P. 1996. An activated form of Notch    influences the choice between CD4 and CD8 T cell lineages. Cell 87,    483-492.-   Rosenbluth, J. 1995. Glial membranes and axoglial junctions. In:    Neuroglia (Kettenmann, H. and Ransom, B. R. eds). Oxford University    Press, New York. pp 613-633.-   Sakurai, T., Lustig, M., Nativ, M., Hemperly, J. J., Schlessinger,    J., Peles, E., and Grumet, M. 1997. Induction of neurite outgrowth    through contactin and Nr-CAM by extracellular regions of glial    receptor tyrosine phosphatase beta. J. Cell Biol. 136, 907-918.-   Salzer, J. L. 1997. Clustering sodium channels at the node of    Ranvier: Close encounters of the axon-glia kind. Neuron 18, 843-846.-   Sauvageot, C. M. & Stiles, C. D. 2002. Curr. Opin. Neurobiol. 12,    244-249.-   Schnaldelbach, O., Ozen, I., Blashuk, O. W., Gour, B. J.,    Meyer, R. L. and Fawcett, J. W. 2001. N-Cadherin is involved in    axon-oligodendrocyte contact and myelination. Mol. Cell. Neurosci.    17, 1084-1093.-   Schroeter, E. H., Kisslinger, J. A., and Kopan, R. 1998. Notch-1    signalling requires ligand-induced proteolytic release of    intracellular domain. Nature 393, 382-386.-   Schwab, M. E. and Schnell, L. 1989. Region-specific appearance of    myelin constituents in the developing rat spinal cord. J.    Neurocytol. 18, 161-169.-   Shimazaki, K., Hosoya, H., Takeda, Y., Kobayashi, S., and    Watanabe, K. 1998. Age-related decline of F3/contactin in rat    hippocampus. Neurosci. Lett. 245, 117-20.-   Shimizu, K., Chiba, S., Kumano, K., Hosoya, N., Takahashi, T.,    Kanda, Y., Hamada, Y., Yazaki, Y., and Hirai, H. 1999. Mouse jagged1    physically interacts with notch2 and other notch receptors.    Assessment by quantitative methods. J. Biol. Chem. 274, 32961-32969.-   Small, D., Kovalenko, D., Kacer, D., Liaw, L., Landriscina, M., Di    Serio, C., Prudovsky, I., and Maciag, T. 2001. Soluble Jagged 1    represses the function of its transmembrane form to induce the    formation of the Src-dependent chord-like phenotype. J. Biol. Chem.    276, 32022-32030.-   Tait, S., Gunn-Moore, F., Collinson, J. M., Huang, J., Lubetzki, C.,    Pedraza, L., Sherman, D. L., Colman, D. R. and Brophy P. J. 2000. An    oligodendrocyte cell adhesion molecule at the site of assembly of    the paranodal axo-glial junction. J. Cell Biol. 150, 657-666.-   Tanigaki, K., Nogaki, F., Takahashi, J., Tashiro, K., Kurooka, H.,    and Honjo, T. 2001. Notch1 and Notch3 instructively restrict    bFGF-responsive multipotent neural progenitor cells to an astroglial    fate. Neuron 29, 45-55.-   Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic    transfer of proteins from polyacrylamide gels to nitrocellulose    sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA    76, 4350-4354.-   Tsiotra, P. C., Karagogeos, D., Theodorakis, K., Michaelidis, T. M.,    Modi, W. S., Furley, A. J., Jessel, T. M. and    Papamatheakis, J. 1993. Isolation of the cDNA and chromosomal    localization of the gene (TAX1) encoding the human axonal    glycoprotein TAG-1. Genomics 18, 562-567.-   Umemori, H., Sato, S., Yagi, T., Aizawa, S., and Yamamoto, T. 1994.    Initial events of myelination involve Fyn tyrosine kinase    signalling. Nature 367, 572-576.-   Wakamatsu, Y., Maynard, T. M., and Weston, J. A. 2000. Fate    determination of neural crest cells by NOTCH-mediated lateral    inhibition and asymmetrical cell division during gangliogenesis.    Development 127, 2811-2821.-   Wang, S., Sdrulla, A. D., diSibio, G., Bush, G., Nofziger, D.,    Hicks, C., Weinmaster, G. and Barres, B. A. 1998. Notch receptor    activation inhibits oligodendrocyte differentiation. Neuron 21,    63-75.-   Ward, R. E., Lamb, R. S. and Fehon, R. G. 1998. A conserved    functional domain of drosophila coracle is required for localization    at the septate junction and has membrane-organizing activity. J.    Cell Biol. 140, 1463-1473.-   Weinmaster, G. 2000. Notch signal transduction: a real rip and more.    Curr. Opin. Gen. Dev. 10, 363-369.-   Wintergerst, E. S., Fuss, B. and Bartsch, U. 1993. Localization of    janusin mRNA in the central nervous system of the developing and    adult mouse. Eur. J. Neurosci. 5, 299-310.-   Xiao, Z. C., Taylor, J., Montag, D., Rougon, G. and    Schachner, M. 1996. Distinct effects of tenascin-R domains in    neuronal cell functions and identification of the domain interacting    with the neuronal recognition molecule F3/11. Eur. J. Neurosci. 8,    766-782.-   Xiao, Z. C., Bartsch, U., Margolis, R. K., Rougon, G., Montag, D.,    and Schachner M. 1997. Isolation of a tenascin-R binding protein    from mouse brain membranes. A phosphacan-related chondroitin sulfate    proteoglycan. J. Biol. Chem. 272, 32092-32101.-   Xiao, Z. C., Hillenbrand, R., Schachner, M., Thermes, S., Rougon, G.    and Gomez, S. 1997. Signalling events following the interaction of    the neuronal adhesion molecule F3 with the N-terminal domain of    tenascin-R. J. Neurosci. Res. 49, 698-709.-   Xiao, Z. C., Revest, J. M., Laeng, P., Rougon, G., Schachner, M. and    Montag, D. 1998. Defasciculation of neurites is mediated by    tenascin-R and its neuronal receptor F3/11. J. Neurosci. Res. 52,    390-404.-   Yamamoto, N., Yamamoto, S., Inagaki, F., Kawaichi, M., Fukamizu, A.,    Kishi, N., Matsuno, K., Nakamura, K., Weinmaster, G., Okano, H., and    Nakafuku, M. 2001. Role of Deltex-1 as a transcriptional regulator    downstream of the Notch receptor. J. Biol. Chem. 276, 45031-45040.-   Yang, H., Xiao, Z. C., Becker, B., Hillenbrand, R., Rougon, G. and    Schachner, M. 1999. Role for myelin-associated glycoprotein as a    functional tenascin-R receptor. J. Neurosci. Res. 55, 687-701.-   Zeng, L., D'Alessandri, L., Kalousek, M. B., Vaughan, L. and    Pallen, C. J. 1999. Protein tyrosine phosphatase alpha (PTPα) and    contactin form a novel neuronal receptor complex linked to the    intracellular tyrosine kinase fyn. J. Cell Biol. 147, 707-714.

1. A composition comprising Nogo and Caspr, or mimetics thereof, or a substance capable of promoting interaction between Nogo and Caspr, in combination with a carrier.
 2. A composition according to claim 1 wherein the composition comprises a complex between Nogo and Caspr, or a mimetic of said complex.
 3. A composition according to claim 1 comprising Nogo-66.
 4. A composition according to claim 1 comprising Caspr1.
 5. A composition according to claim 1 wherein the substance capable of promoting interaction between Nogo and Caspr is an antibody.
 6. A composition according to claim 5 wherein the antibody is capable of binding to both Nogo and Caspr.
 7. A composition according to claim 1, which is a pharmaceutical composition.
 8. A pharmaceutical composition according to claim 7 which is formulated for injection in vivo.
 9. A pharmaceutical composition according to claim 8 which is formulated for direct injection into the CNS. 10.-15. (canceled)
 16. A method of stimulating myelination of a neural axon, comprising contacting a neuron or an oligodendroglial cell with a composition according to claim
 1. 17. A method of treating a subject having disease of, or injury to, the central nervous system, comprising administering to the subject a pharmaceutical composition according to claim
 7. 18. A method according to claim 17 wherein the subject has SCI, MS, epilepsy or stroke.
 19. A method of screening for a substance capable of modulating interaction between Nogo and Caspr, the method comprising contacting Nogo, Caspr and a candidate substance, and determining the interaction between Nogo and Caspr.
 20. A method according to claim 19 further comprising contacting Nogo and Caspr in the absence of said candidate substance under otherwise analogous conditions, and determining the interaction between Nogo and Caspr.
 21. A method according to claim 19 comprising contacting a complex between Nogo and Caspr with the candidate substance.
 22. A method according to claim 19 wherein one of Nogo and Caspr is present in or on a cell.
 23. A method according to claim 22 wherein said one of Nogo and Caspr is expressed from a vector introduced into said cell.
 24. A method according to claim 19 wherein one of Nogo and Caspr is immobilised on a solid support.
 25. A method of manufacturing a pharmaceutical formulation comprising, having identified a substance capable of modulating interaction between Nogo and Caspr by a method according to claim 19, the further step of formulating said substance with a pharmaceutically acceptable carrier.
 26. A method according to claim 25 comprising the further step of optimising said substance for administration in vivo. 27.-62. (canceled) 